Plant promoter for transgene expression

ABSTRACT

This disclosure concerns compositions and methods for promoting transcription of a nucleotide sequence in a plant or plant cell, employing a promoter from a GmTEFs1 gene. Some embodiments relate to a promoter or a 5′ UTR from a GmTEFs1 gene that functions in plants to promote transcription of operably linked nucleotide sequences. Other embodiments relate to a 3′ UTR or a terminator from a GmTEFs1 gene that functions in plants to promote transcription of operably linked nucleotide sequences.

INCORPORATION BY REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/587,034 filed Nov. 16, 2017 which is expresslyincorporated by reference in its entirety herein.

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 30.0 KB ASCII (Text) file named“80596_ST25” created on Nov. 16, 2017.

BACKGROUND

Many plant species are capable of being transformed with transgenes tointroduce agronomically desirable traits or characteristics. Theresulting plant species are developed and/or modified to have particulardesirable traits. Generally, desirable traits include, for example,improving nutritional value quality, increasing yield, conferring pestor disease resistance, increasing drought and stress tolerance,improving horticultural qualities (e.g., pigmentation and growth),imparting herbicide tolerance, enabling the production of industriallyuseful compounds and/or materials from the plant, and/or enabling theproduction of pharmaceuticals.

Transgenic plant species comprising multiple transgenes stacked at asingle genomic locus are produced via plant transformation technologies.Plant transformation technologies result in the introduction of atransgene into a plant cell, recovery of a fertile transgenic plant thatcontains the stably integrated copy of the transgene in the plantgenome, and subsequent transgene expression via transcription andtranslation results in transgenic plants that possess desirable traitsand phenotypes. However, novel gene regulatory elements that allow theproduction of transgenic plant species to highly express multipletransgenes engineered as a trait stack are desirable.

Likewise, novel gene regulatory elements that allow the expression of atransgene within particular tissues or organs of a plant are desirable.For example, increased resistance of a plant to infection by soil-bornepathogens might be accomplished by transforming the plant genome with apathogen-resistance gene such that pathogen-resistance protein isrobustly expressed within the roots of the plant. Alternatively, it maybe desirable to express a transgene in plant tissues that are in aparticular growth or developmental phase such as, for example, celldivision or elongation. Furthermore, it may be desirable to express atransgene in leaf and stem tissues of a plant to provide toleranceagainst herbicides, or resistance against above ground insects andpests.

Therefore, a need exists for new gene regulatory elements that can drivethe desired levels of expression of transgenes in specific planttissues.

BRIEF SUMMARY

In embodiments of the subject disclosure, the disclosure relates to anucleic acid vector comprising a promoter operably linked to: apolylinker sequence; a non-GmTEFs1 heterologous coding sequence; whereinsaid promoter comprises a polynucleotide sequence that has at least 95%sequence identity with SEQ ID NO:2. In further embodiments, saidpromoter is 371 bp in length. In other embodiments, said promoterconsists of a polynucleotide sequence that has at least 95% sequenceidentity with SEQ ID NO:2. In additional embodiments, said promoter isoperably linked to a heterologous coding sequence. Accordingly, theheterologous coding sequence encodes a selectable marker protein, aninsecticidal resistance protein, a herbicide tolerance protein, anitrogen use efficiency protein, a water use efficiency protein, a smallRNA molecule, a nutritional quality protein, or a DNA binding protein.In other embodiments, the nucleic acid vector comprises a terminatorpolynucleotide sequence. In additional embodiments, the nucleic acidvector comprises a 3′ untranslated polynucleotide sequence. Inadditional embodiments, the nucleic acid vector comprises a 5′untranslated polynucleotide sequence. In additional embodiments, thenucleic acid vector comprises an intron sequence. In additionalembodiments, said promoter has tissue constitutive expression. Infurther embodiments, the nucleic acid vector comprises a polynucleotidesequence that has at least 95% sequence identity with SEQ ID NO:2operably linked to a heterologous coding sequence. In furtherembodiments, said plant is selected from the group consisting of Zeamays, wheat, rice, sorghum, oats, rye, bananas, sugar cane, Glycine max,cotton, Arabidopsis, tobacco, sunflower, and canola. In yet anotherembedment, said plant is Glycine max. In some embodiments, theheterologous coding sequence is inserted into the genome of said plant.In other embodiments, the promoter comprises a polynucleotide sequencehaving at least 95% sequence identity with SEQ ID NO:2 and said promoteris operably linked to a heterologous coding sequence. In additionalembodiments, the transgenic plant comprises a 3′ untranslated sequence.In further embodiments, said heterologous coding sequence has tissueconstitutive expression. In additional embodiments, the transgenic plantcomprises said promoter of 371 bp in length.

In embodiments of the subject disclosure, the disclosure relates to amethod for producing a transgenic plant cell, the method comprising thesteps of transforming a plant cell with a gene expression cassettecomprising a GmTEFs1 promoter operably linked to at least onepolynucleotide sequence of interest; isolating the transformed plantcell comprising the gene expression cassette; and, producing atransgenic plant cell comprising the GmTEFs1 promoter operably linked toat least one polynucleotide sequence of interest. In other embodiments,the transformation of a plant cell is performed with a planttransformation method. In some aspects, the plant transformation methodis selected from the group consisting of an Agrobacterium-mediatedtransformation method, a biolistics transformation method, a siliconcarbide transformation method, a protoplast transformation method, and aliposome transformation method. In further embodiments, thepolynucleotide sequence of interest is expressed in a plant cell. Inother embodiments, the polynucleotide sequence of interest is stablyintegrated into the genome of the transgenic plant cell. In furtherembodiments, the method comprises regenerating the transgenic plant cellinto a transgenic plant; and, obtaining the transgenic plant, whereinthe transgenic plant comprising the gene expression cassette comprisingthe GmTEFs1 promoter of operably linked to at least one polynucleotidesequence of interest. In other embodiments, the transgenic plant cell isa monocotyledonous transgenic plant cell or a dicotyledonous transgenicplant cell. Examples of a dicotyledonous transgenic plant cell includesan Arabidopsis plant cell, a tobacco plant cell, a Glycine max plantcell, a canola plant cell, and a cotton plant cell. Examples of amonocotyledonous transgenic plant cell includes a Zea mays plant cell, arice plant cell, and a wheat plant cell. In some embodiments, theGmTEFs1 promoter comprises the polynucleotide of SEQ ID NO:2. In otherembodiments, the GmTEFs1 promoter comprises a first polynucleotidesequence of interest operably linked to the 3′ end of SEQ ID NO:2. Inadditional embodiments, the method comprises introducing into the plantcell a polynucleotide sequence of interest operably linked to a GmTEFs1promoter. In further embodiments, the polynucleotide sequence ofinterest operably linked to the GmTEFs1 promoter is introduced into theplant cell by a plant transformation method. Examples of planttransformation methods include Agrobacterium-mediated transformationmethod, a biolistics transformation method, a silicon carbidetransformation method, a protoplast transformation method, and aliposome transformation method. In further embodiments, thepolynucleotide sequence of interest is expressed in embryonic celltissue. In additional embodiments, the polynucleotide sequence ofinterest is stably integrated into the genome of the plant cell. In someembodiments, the transgenic plant cell is a monocotyledonous plant cellor a dicotyledonous plant cell. Examples of dicotyledonous plant cellsinclude an Arabidopsis plant cell, a tobacco plant cell, a Glycine maxplant cell, a canola plant cell, and a cotton plant cell. Examples ofmonocotyledonous plant cells include a Zea mays plant cell, a rice plantcell, and a wheat plant cell.

In embodiments of the subject disclosure, the disclosure relates to atransgenic plant cell comprising a GmTEFs1 promoter. In otherembodiments, the transgenic plant cell comprises a transgenic event. Infurther embodiments, the transgenic event comprises an agronomic trait.Examples of agronomic traits include an insecticidal resistance trait,herbicide tolerance trait, nitrogen use efficiency trait, water useefficiency trait, nutritional quality trait, DNA binding trait,selectable marker trait, small RNA trait, or any combination thereof. Infurther embodiments, the agronomic trait comprises an herbicide toleranttrait. In an aspect of this embodiment, the herbicide tolerant traitcomprises an aad-1 coding sequence. In yet another embodiment, thetransgenic plant cell produces a commodity product. Examples of acommodity product includes protein concentrate, protein isolate, grain,meal, flour, oil, or fiber. In further embodiments, the transgenic plantcell is selected from the group consisting of a dicotyledonous plantcell or a monocotyledonous plant cell. For example, the dicotyledonousplant cell is a Glycine max plant cell. In additional embodiments, theGmTEFs1 promoter comprises a polynucleotide with at least 95% sequenceidentity to the polynucleotide of SEQ ID NO:2. In other embodiments, theGmTEFs1 promoter is 371 bp in length. In some embodiments, the GmTEFs1promoter consists of SEQ ID NO:2. In subsequent embodiments, the GmTEFs1promoter comprises a first polynucleotide sequence of interest operablylinked to the 3′ end of SEQ ID NO:2. In other embodiments, the agronomictrait is expressed in plant tissues. In further embodiments, theisolated polynucleotide comprises a nucleic acid sequence with at least95% sequence identity to the polynucleotide of SEQ ID NO:2. Inadditional embodiments, the isolated polynucleotide drives constitutivetissue expression. In other embodiments, the isolated polynucleotidecomprises expression activity within a plant cell. In some embodiments,the isolated polynucleotide comprise an open-reading framepolynucleotide coding for a polypeptide; and a termination sequence. Insubsequent embodiments, the polynucleotide of SEQ ID NO:2 is 371 bp inlength.

In embodiments of the subject disclosure, the disclosure relates to agene expression cassette comprising a promoter operably linked to aheterologous coding sequence, wherein the promoter comprises apolynucleotide comprising a sequence identity of at least 95% to SEQ IDNO:2. In some embodiments, the polynucleotide has at least 95% sequenceidentity to SEQ ID NO:2. In additional embodiments, the gene expressioncassette comprises an intron. In further embodiments, the geneexpression cassette comprises a 5′ UTR. In subsequent embodiments, thepromoter has constitutive tissue expression. In other embodiments, thepromoter is operably linked to a heterologous coding sequence thatencodes a polypeptide or a small RNA gene. Examples of the encodedpolypeptide or small RNA gene include a heterologous coding sequenceconferring insecticidal resistance, herbicide tolerance, a nucleic acidconferring nitrogen use efficiency, a nucleic acid conferring water useefficiency, a nucleic acid conferring nutritional quality, a nucleicacid encoding a DNA binding protein, and a nucleic acid encoding aselectable marker. In additional embodiments, the gene expressioncassette comprises a 3′ untranslated region. For example, the 3′untranslated region has at least 95% sequence identity to SEQ ID NO:4.In additional embodiments, the gene expression cassette comprises a 5′untranslated region. For example, the 5′ untranslated region has atleast 95% sequence identity to SEQ ID NO:3. In additional embodiments,the gene expression cassette comprises a terminator region. For example,the terminator region has at least 95% sequence identity to SEQ ID NO:5.In other embodiments the subject disclosure relates to a recombinantvector comprising the gene expression cassette, wherein the vector isselected from the group consisting of a plasmid, a cosmid, a bacterialartificial chromosome, a virus, and a bacteriophage. In otherembodiments the subject disclosure relates to a transgenic cellcomprising the gene expression cassette. In an aspect of thisembodiment, the transgenic cell is a transgenic plant cell. In otheraspects of this embodiment the transgenic plant comprises the transgenicplant cell. In further aspects the transgenic plant is amonocotyledonous plant or dicotyledonous plant. Examples of amonocotyledonous plant is include a maize plant, a rice plant, and awheat plant. In further aspects of the embodiment, the transgenic plantproduces a seed comprising the gene expression cassette. In otherembodiments, the promoter is a constitutive tissue promoter. In someembodiments, the promoter is a constitutive promoter.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Provides a FIGURE of a linear synthetic DNA fragment containingGmTEFs1 promoter, 5′ UTR and terminator linked by the multiple cloningsite.

DETAILED DESCRIPTION I. Overview of Several Embodiments

Development of transgenic plant products is becoming increasinglycomplex. Commercially viable transgenic plants now require the stackingof multiple transgenes into a single locus. Plant promoters and 3′UTRs/terminators used for basic research or biotechnologicalapplications are generally unidirectional, directing only one gene thathas been fused at its 3′ end (downstream) for the promoter, or at its 5′end (upstream) for the 3′ UTR/terminator. Accordingly, eachtransgene/heterologous coding sequence usually requires a promoter and3′ UTR/terminator for expression, wherein multiple regulatory elementsare required to express multiple transgenes within one gene stack. Withan increasing number of transgenes in gene stacks, the same promoterand/or 3′ UTR/terminator is routinely used to obtain optimal levels ofexpression patterns of different transgenes. Obtaining optimal levels oftransgene/heterologous coding sequence expression is necessary for theproduction of a single polygenic trait. Unfortunately, multi-geneconstructs driven by the same promoter and/or 3′ UTR/terminator areknown to cause gene silencing resulting in less efficacious transgenicproducts in the field. The repeated promoter and/or 3′ UTR/terminatorelements may lead to homology-based gene silencing. In addition,repetitive sequences within a transgene/heterologous coding sequence maylead to gene intra locus homologous recombination resulting inpolynucleotide rearrangements. The silencing and rearrangement oftransgenes will likely have an undesirable affect on the performance ofa transgenic plant produced to express transgenes. Further, excess oftranscription factor (TF)-binding sites due to promoter repetition cancause depletion of endogenous TFs leading to transcriptionalinactivation. Given the need to introduce multiple genes into plants formetabolic engineering and trait stacking, a variety of promoters and/or3′ UTRs/terminators are required to develop transgenic crops that drivethe expression of multiple genes.

A particular problem in promoter and/or 3′ UTR/terminator identificationis the need to identify tissue-specific/preferred promoters, related tospecific cell types, developmental stages and/or functions in the plantthat are not expressed in other plant tissues. Tissue specific (i.e.,tissue preferred) or organ specific promoters drive gene expression in acertain tissue such as in the kernel, root, leaf, or tapetum of theplant. Tissue and developmental stage specific promoters and/or 3′UTRs/terminators can be initially identified from observing theexpression of genes, which are expressed in particular tissues or atparticular time periods during plant development. These tissuespecific/preferred promoters and/or 3′ UTRs/terminators are required forcertain applications in the transgenic plant industry and are desirableas they permit specific expression of heterologous genes in a tissueand/or developmental stage selective manner, indicating expression ofthe heterologous gene differentially at various organs, tissues and/ortimes, but not in other undesirable tissues. For example, increasedresistance of a plant to infection by soil-borne pathogens might beaccomplished by transforming the plant genome with a pathogen-resistancegene such that pathogen-resistance protein is robustly expressed withinthe roots of the plant. Alternatively, it may be desirable to express atransgene/heterologous coding sequence in plant tissues that are in aparticular growth or developmental phase such as, for example, celldivision or elongation. Another application is the desirability of usingtissue specific/preferred promoters and/or 3′ UTRs/terminators toconfine the expression of the transgenes encoding an agronomic trait inspecific tissues types like developing parenchyma cells. As such, aparticular problem in the identification of promoters and/or 3′UTRs/terminators is how to identify the promoters, and to relate theidentified promoter to developmental properties of the cell forspecific/preferred tissue expression.

Another problem regarding the identification of a promoter is therequirement to clone all relevant cis-acting and trans-activatingtranscriptional control elements so that the cloned DNA fragment drivestranscription in the wanted specific expression pattern. Given that suchcontrol elements are located distally from the translation initiation orstart site, the size of the polynucleotide that is selected to comprisethe promoter is of importance for providing the level of expression andthe expression patterns of the promoter polynucleotide sequence. It isknown that promoter lengths include functional information, anddifferent genes have been shown to have promoters longer or shorter thanpromoters of the other genes in the genome. Elucidating thetranscription start site of a promoter and predicting the functionalgene elements in the promoter region is challenging. Further adding tothe challenge are the complexity, diversity and inherent degeneratenature of regulatory motifs and cis- and trans-regulatory elements(Blanchette, Mathieu, et al. “Genome-wide computational prediction oftranscriptional regulatory modules reveals new insights into human geneexpression.” Genome research 16.5 (2006): 656-668). The cis- andtrans-regulatory elements are located in the distal parts of thepromoter which regulate the spatial and temporal expression of a gene tooccur only at required sites and at specific times (Porto, Milena Silva,et al. “Plant promoters: an approach of structure and function.”Molecular biotechnology 56.1 (2014): 38-49). Accordingly, theidentification of promoter regulatory elements requires that anappropriate sequence of a specific size containing the necessary cis-and trans-regulatory elements is obtained that will result in drivingexpression of an operably linked transgene/heterologous coding sequencein a desirable manner.

Provided are methods and compositions for overcoming such problemsthrough the use of GmTEFs1 gene regulatory elements to expresstransgenes in planta.

II. Terms and Abbreviations

Throughout the application, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

As used herein, the articles, “a,” “an,” and “the” include pluralreferences unless the context clearly and unambiguously dictatesotherwise.

The term “isolated”, as used herein means having been removed from itsnatural environment, or removed from other compounds present when thecompound is first formed. The term “isolated” embraces materialsisolated from natural sources as well as materials (e.g., nucleic acidsand proteins) recovered after preparation by recombinant expression in ahost cell, or chemically-synthesized compounds such as nucleic acidmolecules, proteins, and peptides.

The term “purified”, as used herein relates to the isolation of amolecule or compound in a form that is substantially free ofcontaminants normally associated with the molecule or compound in anative or natural environment, or substantially enriched inconcentration relative to other compounds present when the compound isfirst formed, and means having been increased in purity as a result ofbeing separated from other components of the original composition. Theterm “purified nucleic acid” is used herein to describe a nucleic acidsequence which has been separated, produced apart from, or purified awayfrom other biological compounds including, but not limited topolypeptides, lipids and carbohydrates, while effecting a chemical orfunctional change in the component (e.g., a nucleic acid may be purifiedfrom a chromosome by removing protein contaminants and breaking chemicalbonds connecting the nucleic acid to the remaining DNA in thechromosome).

The term “synthetic”, as used herein refers to a polynucleotide (i.e., aDNA or RNA) molecule that was created via chemical synthesis as an invitro process. For example, a synthetic DNA may be created during areaction within an Eppendorf™ tube, such that the synthetic DNA isenzymatically produced from a native strand of DNA or RNA. Otherlaboratory methods may be utilized to synthesize a polynucleotidesequence. Oligonucleotides may be chemically synthesized on an oligosynthesizer via solid-phase synthesis using phosphoramidites. Thesynthesized oligonucleotides may be annealed to one another as acomplex, thereby producing a “synthetic” polynucleotide. Other methodsfor chemically synthesizing a polynucleotide are known in the art, andcan be readily implemented for use in the present disclosure.

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

For the purposes of the present disclosure, a “gene,” includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites, introns and locus control regions.

As used herein the terms “native” or “natural” define a condition foundin nature. A “native DNA sequence” is a DNA sequence present in naturethat was produced by natural means or traditional breeding techniquesbut not generated by genetic engineering (e.g., using molecularbiology/transformation techniques).

As used herein a “transgene” is defined to be a nucleic acid sequencethat encodes a gene product, including for example, but not limited to,an mRNA. In one embodiment the transgene/heterologous coding sequence isan exogenous nucleic acid, where the transgene/heterologous codingsequence has been introduced into a host cell by genetic engineering (orthe progeny thereof) where the transgene/heterologous coding sequence isnot normally found. In one example, a transgene/heterologous codingsequence encodes an industrially or pharmaceutically useful compound, ora gene encoding a desirable agricultural trait (e.g., anherbicide-resistance gene). In yet another example, atransgene/heterologous coding sequence is an antisense nucleic acidsequence, wherein expression of the antisense nucleic acid sequenceinhibits expression of a target nucleic acid sequence. In one embodimentthe transgene/heterologous coding sequence is an endogenous nucleicacid, wherein additional genomic copies of the endogenous nucleic acidare desired, or a nucleic acid that is in the antisense orientation withrespect to the sequence of a target nucleic acid in a host organism.

As used herein the term “non-GmTEFs1 transgene” or “non-GmTEFs1 gene” isany transgene/heterologous coding sequence that has less than 80%sequence identity with the GmTEFs1 gene coding sequence.

As used herein, “heterologous DNA coding sequence” means any codingsequence other than the one that naturally encodes the GmTEFs1 gene, orany homolog of the expressed GmTEFs1 protein. The term “heterologous” isused in the context of this invention for any combination of nucleicacid sequences that is not normally found intimately associated innature.

A “gene product” as defined herein is any product produced by the gene.For example the gene product can be the direct transcriptional productof a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA,ribozyme, structural RNA or any other type of RNA) or a protein producedby translation of a mRNA. Gene products also include RNAs which aremodified, by processes such as capping, polyadenylation, methylation,and editing, and proteins modified by, for example, methylation,acetylation, phosphorylation, ubiquitination, ADP-ribosylation,myristilation, and glycosylation. Gene expression can be influenced byexternal signals, for example, exposure of a cell, tissue, or organismto an agent that increases or decreases gene expression. Expression of agene can also be regulated anywhere in the pathway from DNA to RNA toprotein. Regulation of gene expression occurs, for example, throughcontrols acting on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization, or degradationof specific protein molecules after they have been made, or bycombinations thereof. Gene expression can be measured at the RNA levelor the protein level by any method known in the art, including, withoutlimitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ,or in vivo protein activity assay(s).

As used herein the term “gene expression” relates to the process bywhich the coded information of a nucleic acid transcriptional unit(including, e.g., genomic DNA) is converted into an operational,non-operational, or structural part of a cell, often including thesynthesis of a protein. Gene expression can be influenced by externalsignals; for example, exposure of a cell, tissue, or organism to anagent that increases or decreases gene expression. Expression of a genecan also be regulated anywhere in the pathway from DNA to RNA toprotein. Regulation of gene expression occurs, for example, throughcontrols acting on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization, or degradationof specific protein molecules after they have been made, or bycombinations thereof. Gene expression can be measured at the RNA levelor the protein level by any method known in the art, including, withoutlimitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ,or in vivo protein activity assay(s).

As used herein, “homology-based gene silencing” (HBGS) is a generic termthat includes both transcriptional gene silencing andpost-transcriptional gene silencing. Silencing of a target locus by anunlinked silencing locus can result from transcription inhibition(transcriptional gene silencing; TGS) or mRNA degradation(post-transcriptional gene silencing; PTGS), owing to the production ofdouble-stranded RNA (dsRNA) corresponding to promoter or transcribedsequences, respectively. The involvement of distinct cellular componentsin each process suggests that dsRNA-induced TGS and PTGS likely resultfrom the diversification of an ancient common mechanism. However, astrict comparison of TGS and PTGS has been difficult to achieve becauseit generally relies on the analysis of distinct silencing loci. In someinstances, a single transgene locus can triggers both TGS and PTGS,owing to the production of dsRNA corresponding to promoter andtranscribed sequences of different target genes. Mourrain et al. (2007)Planta 225:365-79. It is likely that siRNAs are the actual moleculesthat trigger TGS and PTGS on homologous sequences: the siRNAs would inthis model trigger silencing and methylation of homologous sequences incis and in trans through the spreading of methylation of transgenesequences into the endogenous promoter.

As used herein, the term “nucleic acid molecule” (or “nucleic acid” or“polynucleotide”) may refer to a polymeric form of nucleotides, whichmay include both sense and anti-sense strands of RNA, cDNA, genomic DNA,and synthetic forms and mixed polymers of the above. A nucleotide mayrefer to a ribonucleotide, deoxyribonucleotide, or a modified form ofeither type of nucleotide. A “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide”. A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term may refer to a molecule of RNA or DNA ofindeterminate length. The term includes single- and double-strandedforms of DNA. A nucleic acid molecule may include either or bothnaturally-occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications (e.g., uncharged linkages: for example, methylphosphonates, phosphotriesters, phosphoramidites, carbamates, etc.;charged linkages: for example, phosphorothioates, phosphorodithioates,etc.; pendent moieties: for example, peptides; intercalators: forexample, acridine, psoralen, etc.; chelators; alkylators; and modifiedlinkages: for example, alpha anomeric nucleic acids, etc.). The term“nucleic acid molecule” also includes any topological conformation,including single-stranded, double-stranded, partially duplexed,triplexed, hairpinned, circular, and padlocked conformations.

Transcription proceeds in a 5′ to 3′ manner along a DNA strand. Thismeans that RNA is made by the sequential addition ofribonucleotide-5′-triphosphates to the 3′ terminus of the growing chain(with a requisite elimination of the pyrophosphate). In either a linearor circular nucleic acid molecule, discrete elements (e.g., particularnucleotide sequences) may be referred to as being “upstream” or “5′”relative to a further element if they are bonded or would be bonded tothe same nucleic acid in the 5′ direction from that element. Similarly,discrete elements may be “downstream” or “3′” relative to a furtherelement if they are or would be bonded to the same nucleic acid in the3′ direction from that element.

A base “position”, as used herein, refers to the location of a givenbase or nucleotide residue within a designated nucleic acid. Thedesignated nucleic acid may be defined by alignment (see below) with areference nucleic acid.

Hybridization relates to the binding of two polynucleotide strands viaHydrogen bonds. Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidmolecules consist of nitrogenous bases that are either pyrimidines(cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) andguanine (G)). These nitrogenous bases form hydrogen bonds between apyrimidine and a purine, and the bonding of the pyrimidine to the purineis referred to as “base pairing.” More specifically, A will hydrogenbond to T or U, and G will bond to C. “Complementary” refers to the basepairing that occurs between two distinct nucleic acid sequences or twodistinct regions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. The oligonucleotide need not be 100% complementary to itstarget sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget DNA or RNA molecule interferes with the normal function of thetarget DNA or RNA, and there is sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the chosen hybridization methodand the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na+ and/or Mg2+ concentration) of thehybridization buffer will contribute to the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chs. 9 and 11.

As used herein, “stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 50% mismatch betweenthe hybridization molecule and the DNA target. “Stringent conditions”include further particular levels of stringency. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 50% sequence mismatch will not hybridize; conditions of “highstringency” are those under which sequences with more than 20% mismatchwill not hybridize; and conditions of “very high stringency” are thoseunder which sequences with more than 10% mismatch will not hybridize.

In particular embodiments, stringent conditions can includehybridization at 65° C., followed by washes at 65° C. with 0.1×SSC/0.1%SDS for 40 minutes.

The following are representative, non-limiting hybridization conditions:

-   -   Very High Stringency: Hybridization in 5×SSC buffer at 65° C.        for 16 hours; wash twice in 2×SSC buffer at room temperature for        15 minutes each; and wash twice in 0.5×SSC buffer at 65° C. for        20 minutes each.    -   High Stringency: Hybridization in 5×-6×SSC buffer at 65-70° C.        for 16-20 hours; wash twice in 2×SSC buffer at room temperature        for 5-20 minutes each; and wash twice in 1×SSC buffer at        55-70° C. for 30 minutes each.    -   Moderate Stringency: Hybridization in 6×SSC buffer at room        temperature to 55° C. for 16-20 hours; wash at least twice in        2×-3×SSC buffer at room temperature to 55° C. for 20-30 minutes        each.

In particular embodiments, specifically hybridizable nucleic acidmolecules can remain bound under very high stringency hybridizationconditions. In these and further embodiments, specifically hybridizablenucleic acid molecules can remain bound under high stringencyhybridization conditions. In these and further embodiments, specificallyhybridizable nucleic acid molecules can remain bound under moderatestringency hybridization conditions.

As used herein, the term “oligonucleotide” refers to a short nucleicacid polymer. Oligonucleotides may be formed by cleavage of longernucleic acid segments, or by polymerizing individual nucleotideprecursors. Automated synthesizers allow the synthesis ofoligonucleotides up to several hundred base pairs in length. Becauseoligonucleotides may bind to a complementary nucleotide sequence, theymay be used as probes for detecting DNA or RNA. Oligonucleotidescomposed of DNA (oligodeoxyribonucleotides) may be used in PCR, atechnique for the amplification of small DNA sequences. In PCR, theoligonucleotide is typically referred to as a “primer”, which allows aDNA polymerase to extend the oligonucleotide and replicate thecomplementary strand.

The terms “percent sequence identity” or “percent identity” or“identity” are used interchangeably to refer to a sequence comparisonbased on identical matches between correspondingly identical positionsin the sequences being compared between two or more amino acid ornucleotide sequences. The percent identity refers to the extent to whichtwo optimally aligned polynucleotide or peptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. Hybridization experiments and mathematical algorithms knownin the art may be used to determine percent identity. Many mathematicalalgorithms exist as sequence alignment computer programs known in theart that calculate percent identity. These programs may be categorizedas either global sequence alignment programs or local sequence alignmentprograms.

Global sequence alignment programs calculate the percent identity of twosequences by comparing alignments end-to-end in order to find exactmatches, dividing the number of exact matches by the length of theshorter sequences, and then multiplying by 100. Basically, thepercentage of identical nucleotides in a linear polynucleotide sequenceof a reference (“query) polynucleotide molecule as compared to a test(“subject”) polynucleotide molecule when the two sequences are optimallyaligned (with appropriate nucleotide insertions, deletions, or gaps).

Local sequence alignment programs are similar in their calculation, butonly compare aligned fragments of the sequences rather than utilizing anend-to-end analysis. Local sequence alignment programs such as BLAST canbe used to compare specific regions of two sequences. A BLAST comparisonof two sequences results in an E-value, or expectation value, thatrepresents the number of different alignments with scores equivalent toor better than the raw alignment score, S, that are expected to occur ina database search by chance. The lower the E value, the more significantthe match. Because database size is an element in E-value calculations,E-values obtained by BLASTing against public databases, such as GENBANK,have generally increased over time for any given query/entry match. Insetting criteria for confidence of polypeptide function prediction, a“high” BLAST match is considered herein as having an E-value for the topBLAST hit of less than 1E-30; a medium BLASTX E-value is 1E-30 to 1E-8;and a low BLASTX E-value is greater than 1E-8. The protein functionassignment in the present invention is determined using combinations ofE-values, percent identity, query coverage and hit coverage. Querycoverage refers to the percent of the query sequence that is representedin the BLAST alignment. Hit coverage refers to the percent of thedatabase entry that is represented in the BLAST alignment. In oneembodiment of the invention, function of a query polypeptide is inferredfrom function of a conserved protein sequence where either (1)hit_p<1e-30 or % identity>35% AND query_coverage>50% ANDhit_coverage>50%, or (2) hit_p<1e-8 AND query_coverage>70% ANDhit_coverage>70%. The following abbreviations are produced during aBLAST analysis of a sequence.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described. In anembodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using anAlignX alignment program of the Vector NTI suite (Invitrogen, Carlsbad,Calif.). The AlignX alignment program is a global sequence alignmentprogram for polynucleotides or proteins. In an embodiment, the subjectdisclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the MegAlign program ofthe LASERGENE bioinformatics computing suite (MegAlign™ (©1993-2016).DNASTAR. Madison, Wis.). The MegAlign program is global sequencealignment program for polynucleotides or proteins. In an embodiment, thesubject disclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the Clustal suite ofalignment programs, including, but not limited to, ClustalW and ClustalV(Higgins and Sharp (1988) Gene. December 15; 73(1):237-44; Higgins andSharp (1989) CABIOS 5:151-3; Higgins et al. (1992) Comput. Appl. Biosci.8:189-91). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the GCG suite of programs (Wisconsin Package Version9.0, Genetics Computer Group (GCG), Madison, Wis.). In an embodiment,the subject disclosure relates to calculating percent identity betweentwo polynucleotides or amino acid sequences using the BLAST suite ofalignment programs, for example, but not limited to, BLASTP, BLASTN,BLASTX, etc. (Altschul et al. (1990) J. Mol. Biol. 215:403-10). Furtherexamples of such BLAST alignment programs include Gapped-BLAST orPSI-BLAST (Altschul et al., 1997). In an embodiment, the subjectdisclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the FASTA suite ofalignment programs, including, but not limited to, FASTA, TFASTX,TFASTY, SSEARCH, LALIGN etc. (Pearson (1994) Comput. Methods Genome Res.[Proc. Int. Symp.], Meeting Date 1992 (Suhai and Sandor, Eds.), Plenum:New York, N.Y., pp. 111-20). In an embodiment, the subject disclosurerelates to calculating percent identity between two polynucleotides oramino acid sequences using the T-Coffee alignment program (Notredame,et. al. (2000) J. Mol. Biol. 302, 205-17). In an embodiment, the subjectdisclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the DIALIGN suite ofalignment programs, including, but not limited to DIALIGN, CHAOS,DIALIGN-TX, DIALIGN-T etc. (Al Ait, et. al. (2013) DIALIGN at GOBICSNuc. Acids Research 41, W3-W7). In an embodiment, the subject disclosurerelates to calculating percent identity between two polynucleotides oramino acid sequences using the MUSCLE suite of alignment programs (Edgar(2004) Nucleic Acids Res. 32(5): 1792-1797). In an embodiment, thesubject disclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the MAFFT alignmentprogram (Katoh, et. al. (2002) Nucleic Acids Research 30(14):3059-3066). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the Genoogle program (Albrecht, Felipe. arXiv150702987v1[cs.DC] 10 Jul. 2015). In an embodiment, the subject disclosure relatesto calculating percent identity between two polynucleotides or aminoacid sequences using the HMMER suite of programs (Eddy. (1998)Bioinformatics, 14:755-63). In an embodiment, the subject disclosurerelates to calculating percent identity between two polynucleotides oramino acid sequences using the PLAST suite of alignment programs,including, but not limited to, TPLASTN, PLASTP, KLAST, and PLASTX(Nguyen & Lavenier. (2009) BMC Bioinformatics, 10:329). In anembodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theUSEARCH alignment program (Edgar (2010) Bioinformatics 26(19), 2460-61).In an embodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theSAM suite of alignment programs (Hughey & Krogh (January 1995) TechnicalReport UCSC0CRL-95-7, University of California, Santa Cruz). In anembodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theIDF Searcher (O'Kane, K. C., The Effect of Inverse Document FrequencyWeights on Indexed Sequence Retrieval, Online Journal of Bioinformatics,Volume 6 (2) 162-173, 2005). In an embodiment, the subject disclosurerelates to calculating percent identity between two polynucleotides oramino acid sequences using the Parasail alignment program. (Daily, Jeff.Parasail: SIMD C library for global, semi-global, and local pairwisesequence alignments. BMC Bioinformatics. 17:18. Feb. 10, 2016). In anembodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theScalaBLAST alignment program (Oehmen C, Nieplocha J. “ScalaBLAST: Ascalable implementation of BLAST for high-performance data-intensivebioinformatics analysis.” IEEE Transactions on Parallel & DistributedSystems 17 (8): 740-749 August 2006). In an embodiment, the subjectdisclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the SWIPE alignmentprogram (Rognes, T. Faster Smilth-Waterman database searches withinter-sequence SIMD parallelization. BMC Bioinformatics. 12, 221(2011)). In an embodiment, the subject disclosure relates to calculatingpercent identity between two polynucleotides or amino acid sequencesusing the ACANA alignment program (Weichun Huang, David M. Umbach, andLeping Li, Accurate anchoring alignment of divergent sequences.Bioinformatics 22:29-34, Jan. 1, 2006). In an embodiment, the subjectdisclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the DOTLET alignmentprogram (Junier, T. & Pagni, M. DOTLET: diagonal plots in a web browser.Bioinformatics 16(2): 178-9 Feb. 2000). In an embodiment, the subjectdisclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the G-PAS alignmentprogram (Frohmberg, W., et al. G-PAS 2.0—an improved version of proteinalignment tool with an efficient backtracking routine on multiple GPUs.Bulletin of the Polish Academy of Sciences Technical Sciences, Vol. 60,491 November 2012). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the GapMis alignment program (Flouri, T. et. al., GapMis: A tool for pairwise sequence alignment with a single gap. RecentPat DNA Gene Seq. 7(2): 84-95 August 2013). In an embodiment, thesubject disclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the EMBOSS suite ofalignment programs, including, but not limited to: Matcher, Needle,Stretcher, Water, Wordmatch, etc. (Rice, P., Longden, I. & Bleasby, A.EMBOSS: The European Molecular Biology Open Software Suite. Trends inGenetics 16(6) 276-77 (2000)). In an embodiment, the subject disclosurerelates to calculating percent identity between two polynucleotides oramino acid sequences using the Ngila alignment program (Cartwright, R.Ngila: global pairwise alignments with logarithmic and affine gap costs.Bioinformatics. 23(11): 1427-28. Jun. 1, 2007). In an embodiment, thesubject disclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the probA, also known aspropA, alignment program (Mückstein, U., Hofacker, I L, & Stadler, P F.Stochastic pairwise alignments. Bioinformatics 18 Suppl. 2:S153-60.2002). In an embodiment, the subject disclosure relates to calculatingpercent identity between two polynucleotides or amino acid sequencesusing the SEQALN suite of alignment programs (Hardy, P. & Waterman, M.The Sequence Alignment Software Library at USC. 1997). In an embodiment,the subject disclosure relates to calculating percent identity betweentwo polynucleotides or amino acid sequences using the SIM suite ofalignment programs, including, but not limited to, GAP, NAP, LAP, etc.(Huang, X & Miller, W. A Time-Efficient, Linear-Space Local SimilarityAlgorithm. Advances in Applied Mathematics, vol. 12 (1991) 337-57). Inan embodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theUGENE alignment program (Okonechnikov, K., Golosova, O. & Fursov, M.Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 201228:1166-67). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the BAli-Phy alignment program (Suchard, M A &Redelings, B D. BAli-Phy: simultaneous Bayesian inference of alignmentand phylogeny. Bioinformatics. 22:2047-48. 2006). In an embodiment, thesubject disclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the Base-By-Base alignmentprogram (Brodie, R., et. al. Base-By-Base: Single nucleotide-levelanalysis of whole viral genome alignments, BMC Bioinformatics, 5, 96,2004). In an embodiment, the subject disclosure relates to calculatingpercent identity between two polynucleotides or amino acid sequencesusing the DECIPHER alignment program (ES Wright (2015) “DECIPHER:harnessing local sequence context to improve protein multiple sequencealignment.” BMC Bioinformatics, doi:10.1186/s12859-015-0749-z.). In anembodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theFSA alignment program (Bradley, R K, et. al. (2009) Fast StatisticalAlignment. PLoS Computational Biology. 5:e1000392). In an embodiment,the subject disclosure relates to calculating percent identity betweentwo polynucleotides or amino acid sequences using the Geneious alignmentprogram (Kearse, M., et. al. (2012). Geneious Basic: an integrated andextendable desktop software platform for the organization and analysisof sequence data. Bioinformatics, 28(12), 1647-49). In an embodiment,the subject disclosure relates to calculating percent identity betweentwo polynucleotides or amino acid sequences using the Kalign alignmentprogram (Lassmann, T. & Sonnhammer, E. Kalign—an accurate and fastmultiple sequence alignment algorithm. BMC Bioinformatics 2005 6:298).In an embodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theMAVID alignment program (Bray, N. & Pachter, L. MAVID: ConstrainedAncestral Alignment of Multiple Sequences. Genome Res. 2004 April;14(4): 693-99). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the MSA alignment program (Lipman, D J, et. al. A toolfor multiple sequence alignment. Proc. Nat'l Acad. Sci. USA. 1989;86:4412-15). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the MultAlin alignment program (Corpet, F., Multiplesequence alignment with hierarchical clustering. Nucl. Acids Res., 1988,16(22), 10881-90). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the LAGAN or MLAGAN alignment programs (Brudno, et. al.LAGAN and Multi-LAGAN: efficient tools for large-scale multiplealignment of genomic DNA. Genome Research 2003 April; 13(4): 721-31). Inan embodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theOpal alignment program (Wheeler, T. J., & Kececiouglu, J. D. Multiplealignment by aligning alignments. Proceedings of the 15^(th) ISCBconference on Intelligent Systems for Molecular Biology. Bioinformatics.23, i559-68, 2007). In an embodiment, the subject disclosure relates tocalculating percent identity between two polynucleotides or amino acidsequences using the PicXAA suite of programs, including, but not limitedto, PicXAA, PicXAA-R, PicXAA-Web, etc. (Mohammad, S., Sahraeian, E. &Yoon, B. PicXAA: greedy probabilistic construction of maximum expectedaccuracy alignment of multiple sequences. Nucleic Acids Research.38(15):4917-28. 2010). In an embodiment, the subject disclosure relatesto calculating percent identity between two polynucleotides or aminoacid sequences using the PSAlign alignment program (SZE, S.-H., Lu, Y.,& Yang, Q. (2006) A polynomial time solvable formulation of multiplesequence alignment Journal of Computational Biology, 13, 309-19). In anembodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theStatAlign alignment program (Novák, Á., et. al. (2008) StatAlign: anextendable software package for joint Bayesian estimation of alignmentsand evolutionary trees. Bioinformatics, 24(20):2403-04). In anembodiment, the subject disclosure relates to calculating percentidentity between two polynucleotides or amino acid sequences using theGap alignment program of Needleman and Wunsch (Needleman and Wunsch,Journal of Molecular Biology 48:443-453, 1970). In an embodiment, thesubject disclosure relates to calculating percent identity between twopolynucleotides or amino acid sequences using the BestFit alignmentprogram of Smith and Waterman (Smith and Waterman, Advances in AppliedMathematics, 2:482-489, 1981, Smith et al., Nucleic Acids Research11:2205-2220, 1983). These programs produces biologically meaningfulmultiple sequence alignments of divergent sequences. The calculated bestmatch alignments for the selected sequences are lined up so thatidentities, similarities, and differences can be seen.

The term “similarity” refers to a comparison between amino acidsequences, and takes into account not only identical amino acids incorresponding positions, but also functionally similar amino acids incorresponding positions. Thus similarity between polypeptide sequencesindicates functional similarity, in addition to sequence similarity.

The term “homology” is sometimes used to refer to the level ofsimilarity between two or more nucleic acid or amino acid sequences interms of percent of positional identity (i.e., sequence similarity oridentity). Homology also refers to the concept of evolutionaryrelatedness, often evidenced by similar functional properties amongdifferent nucleic acids or proteins that share similar sequences.

As used herein, the term “variants” means substantially similarsequences. For nucleotide sequences, naturally occurring variants can beidentified with the use of well-known molecular biology techniques, suchas, for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined herein.

For nucleotide sequences, a variant comprises a deletion and/or additionof one or more nucleotides at one or more internal sites within thenative polynucleotide and/or a substitution of one or more nucleotidesat one or more sites in the native polynucleotide. As used herein, a“native” nucleotide sequence comprises a naturally occurring nucleotidesequence. For nucleotide sequences, naturally occurring variants can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis. Generally, variants of aparticular nucleotide sequence of the invention will have at least about40%, 45%, 50%>, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%0, 99% or more sequence identity to thatparticular nucleotide sequence as determined by sequence alignmentprograms and parameters described elsewhere herein. A biologicallyactive variant of a nucleotide sequence of the invention may differ fromthat sequence by as few as 1-15 nucleic acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 nucleic acidresidue.

As used herein the term “operably linked” relates to a first nucleicacid sequence is operably linked with a second nucleic acid sequencewhen the first nucleic acid sequence is in a functional relationshipwith the second nucleic acid sequence. For instance, a promoter isoperably linked with a coding sequence when the promoter affects thetranscription or expression of the coding sequence. When recombinantlyproduced, operably linked nucleic acid sequences are generallycontiguous and, where necessary to join two protein-coding regions, inthe same reading frame. However, elements need not be contiguous to beoperably linked.

As used herein, the term “promoter” refers to a region of DNA thatgenerally is located upstream (towards the 5′ region of a gene) of agene and is needed to initiate and drive transcription of the gene. Apromoter may permit proper activation or repression of a gene that itcontrols. A promoter may contain specific sequences that are recognizedby transcription factors. These factors may bind to a promoter DNAsequence, which results in the recruitment of RNA polymerase, an enzymethat synthesizes RNA from the coding region of the gene. The promotergenerally refers to all gene regulatory elements located upstream of thegene, including, upstream promoters, 5′ UTR, introns, and leadersequences.

As used herein, the term “upstream-promoter” refers to a contiguouspolynucleotide sequence that is sufficient to direct initiation oftranscription. As used herein, an upstream-promoter encompasses the siteof initiation of transcription with several sequence motifs, whichinclude TATA Box, initiator sequence, TFIIB recognition elements andother promoter motifs (Jennifer, E. F. et al., (2002) Genes & Dev., 16:2583-2592). The upstream promoter provides the site of action to RNApolymerase II which is a multi-subunit enzyme with the basal or generaltranscription factors like, TFIIA, B, D, E, F and H. These factorsassemble into a transcription pre initiation complex that catalyzes thesynthesis of RNA from DNA template.

The activation of the upstream-promoter is done by the additionalsequence of regulatory DNA sequence elements to which various proteinsbind and subsequently interact with the transcription initiation complexto activate gene expression. These gene regulatory elements sequencesinteract with specific DNA-binding factors. These sequence motifs maysometimes be referred to as cis-elements. Such cis-elements, to whichtissue-specific or development-specific transcription factors bind,individually or in combination, may determine the spatiotemporalexpression pattern of a promoter at the transcriptional level. Thesecis-elements vary widely in the type of control they exert on operablylinked genes. Some elements act to increase the transcription ofoperably-linked genes in response to environmental responses (e.g.,temperature, moisture, and wounding). Other cis-elements may respond todevelopmental cues (e.g., germination, seed maturation, and flowering)or to spatial information (e.g., tissue specificity). See, for example,Langridge et al., (1989) Proc. Natl. Acad. Sci. USA 86:3219-23. Thesecis-elements are located at a varying distance from transcription startpoint, some cis-elements (called proximal elements) are adjacent to aminimal core promoter region while other elements can be positionedseveral kilobases upstream or downstream of the promoter (enhancers).

As used herein, the terms “5′ untranslated region” or “5′ UTR” isdefined as the untranslated segment in the 5′ terminus of pre-mRNAs ormature mRNAs. For example, on mature mRNAs, a 5′ UTR typically harborson its 5′ end a 7-methylguanosine cap and is involved in many processessuch as splicing, polyadenylation, mRNA export towards the cytoplasm,identification of the 5′ end of the mRNA by the translational machinery,and protection of the mRNAs against degradation.

As used herein, the term “intron” refers to any nucleic acid sequencecomprised in a gene (or expressed polynucleotide sequence of interest)that is transcribed but not translated. Introns include untranslatednucleic acid sequence within an expressed sequence of DNA, as well asthe corresponding sequence in RNA molecules transcribed therefrom. Aconstruct described herein can also contain sequences that enhancetranslation and/or mRNA stability such as introns. An example of onesuch intron is the first intron of gene II of the histone H3 variant ofArabidopsis thaliana or any other commonly known intron sequence.Introns can be used in combination with a promoter sequence to enhancetranslation and/or mRNA stability.

As used herein, the terms “transcription terminator” or “terminator” isdefined as the transcribed segment in the 3′ terminus of pre-mRNAs ormature mRNAs. For example, longer stretches of DNA beyond“polyadenylation signal” site is transcribed as a pre-mRNA. This DNAsequence usually contains transcription termination signal for theproper processing of the pre-mRNA into mature mRNA.

As used herein, the term “3′ untranslated region” or “3′ UTR” is definedas the untranslated segment in a 3′ terminus of the pre-mRNAs or maturemRNAs. For example, on mature mRNAs this region harbors the poly-(A)tail and is known to have many roles in mRNA stability, translationinitiation, and mRNA export. In addition, the 3′ UTR is considered toinclude the polyadenylation signal and transcription terminator.

As used herein, the term “polyadenylation signal” designates a nucleicacid sequence present in mRNA transcripts that allows for transcripts,when in the presence of a poly-(A) polymerase, to be polyadenylated onthe polyadenylation site, for example, located 10 to 30 bases downstreamof the poly-(A) signal. Many polyadenylation signals are known in theart and are useful for the present invention. An exemplary sequenceincludes AAUAAA and variants thereof, as described in Loke J., et al.,(2005) Plant Physiology 138(3); 1457-1468.

A “DNA binding transgene” is a polynucleotide coding sequence thatencodes a DNA binding protein. The DNA binding protein is subsequentlyable to bind to another molecule. A binding protein can bind to, forexample, a DNA molecule (a DNA-binding protein), a RNA molecule (anRNA-binding protein), and/or a protein molecule (a protein-bindingprotein). In the case of a protein-binding protein, it can bind toitself (to form homodimers, homotrimers, etc.) and/or it can bind to oneor more molecules of a different protein or proteins. A binding proteincan have more than one type of binding activity. For example, zincfinger proteins have DNA-binding, RNA-binding, and protein-bindingactivity.

Examples of DNA binding proteins include; meganucleases, zinc fingers,CRISPRs, and TALEN binding domains that can be “engineered” to bind to apredetermined nucleotide sequence. Typically, the engineered DNA bindingproteins (e.g., zinc fingers, CRISPRs, or TALENs) are proteins that arenon-naturally occurring. Non-limiting examples of methods forengineering DNA-binding proteins are design and selection. A designedDNA binding protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP, CRISPR, and/or TALEN designs and bindingdata. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536and WO 03/016496 and U.S. Publication Nos. 20110301073, 20110239315 and20119145940.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP. Zinc finger bindingdomains can be “engineered” to bind to a predetermined nucleotidesequence. Non-limiting examples of methods for engineering zinc fingerproteins are design and selection. A designed zinc finger protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; 6,534,261 and 6,794,136; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496.

In other examples, the DNA-binding domain of one or more of thenucleases comprises a naturally occurring or engineered (non-naturallyoccurring) TAL effector DNA binding domain. See, e.g., U.S. PatentPublication No. 20110301073, incorporated by reference in its entiretyherein. The plant pathogenic bacteria of the genus Xanthomonas are knownto cause many diseases in important crop plants. Pathogenicity ofXanthomonas depends on a conserved type III secretion (T3S) system whichinjects more than different effector proteins into the plant cell. Amongthese injected proteins are transcription activator-like (TALEN)effectors which mimic plant transcriptional activators and manipulatethe plant transcriptome (see Kay et al., (2007) Science 318:648-651).These proteins contain a DNA binding domain and a transcriptionalactivation domain. One of the most well characterized TAL-effectors isAvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al.,(1989) Mol Gen Genet 218: 127-136 and WO2010079430). TAL-effectorscontain a centralized domain of tandem repeats, each repeat containingapproximately 34 amino acids, which are key to the DNA bindingspecificity of these proteins. In addition, they contain a nuclearlocalization sequence and an acidic transcriptional activation domain(for a review see Schornack S, et al., (2006) J Plant Physiol 163(3):256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al., (2007) Appl and Enviro Micro 73(13): 4379-4384).These genes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas. See, e.g., U.S. Patent Publication No.20110301073, incorporated by reference in its entirety.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 bp andthe repeats are typically 91-100% homologous with each other (Bonas etal., ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues at positions 12 and 13 withthe identity of the contiguous nucleotides in the TAL-effector's targetsequence (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch etal., (2009) Science 326:1509-1512). Experimentally, the natural code forDNA recognition of these TAL-effectors has been determined such that anHD sequence at positions 12 and 13 leads to a binding to cytosine (C),NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds toT. These DNA binding repeats have been assembled into proteins with newcombinations and numbers of repeats, to make artificial transcriptionfactors that are able to interact with new sequences and activate theexpression of a non-endogenous reporter gene in plant cells (Boch etal., ibid). Engineered TAL proteins have been linked to a FokI cleavagehalf domain to yield a TAL effector domain nuclease fusion (TALEN)exhibiting activity in a yeast reporter assay (plasmid based target).

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and Archaea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer.” Cas9 cleaves the DNA togenerate blunt ends at the double-stranded break (DSB) at sitesspecified by a 20-nucleotide guide sequence contained within the crRNAtranscript. Cas9 requires both the crRNA and the tracrRNA for sitespecific DNA recognition and cleavage. This system has now beenengineered such that the crRNA and tracrRNA can be combined into onemolecule (the “single guide RNA”), and the crRNA equivalent portion ofthe single guide RNA can be engineered to guide the Cas9 nuclease totarget any desired sequence (see Jinek et al., (2012) Science 337, pp.816-821, Jinek et al., (2013), eLife 2:e00471, and David Segal, (2013)eLife 2:e00563). In other examples, the crRNA associates with thetracrRNA to guide the Cpfl nuclease to a region homologous to the crRNAto cleave DNA with staggered ends (see Zetsche, Bernd, et al. Cell 163.3(2015): 759-771). Thus, the CRISPR/Cas system can be engineered tocreate a DSB at a desired target in a genome, and repair of the DSB canbe influenced by the use of repair inhibitors to cause an increase inerror prone repair.

In other examples, the DNA binding transgene/heterologous codingsequence is a site specific nuclease that comprises an engineered(non-naturally occurring) Meganuclease (also described as a homingendonuclease). The recognition sequences of homing endonucleases ormeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al.,(1997) Nucleic Acids Res. 25:3379-30 3388; Dujon et al., (1989) Gene82:115-118; Perler et al., (1994) Nucleic Acids Res. 22, 11127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al., (1996) J. Mol. Biol.263:163-180; Argast et al., (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier et al.,(2002)Molec. Cell 10:895-905; Epinat et al., (2003) Nucleic Acids Res. 531:2952-2962; Ashworth et al., (2006) Nature 441:656-659; Paques et al.,(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128. The DNA-binding domains of the homing endonucleases andmeganucleases may be altered in the context of the nuclease as a whole(i.e., such that the nuclease includes the cognate cleavage domain) ormay be fused to a heterologous cleavage domain.

As used herein, the term “transformation” encompasses all techniquesthat a nucleic acid molecule can be introduced into such a cell.Examples include, but are not limited to: transfection with viralvectors; transformation with plasmid vectors; electroporation;lipofection; microinjection (Mueller et al., (1978) Cell 15:579-85);Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS™-mediatedtransformation; and microprojectile bombardment. These techniques may beused for both stable transformation and transient transformation of aplant cell. “Stable transformation” refers to the introduction of anucleic acid fragment into a genome of a host organism resulting ingenetically stable inheritance. Once stably transformed, the nucleicacid fragment is stably integrated in the genome of the host organismand any subsequent generation. Host organisms containing the transformednucleic acid fragments are referred to as “transgenic” organisms.“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

An exogenous nucleic acid sequence. In one example, atransgene/heterologous coding sequence is a gene sequence (e.g., anherbicide-resistance gene), a gene encoding an industrially orpharmaceutically useful compound, or a gene encoding a desirableagricultural trait. In yet another example, the transgene/heterologouscoding sequence is an antisense nucleic acid sequence, whereinexpression of the antisense nucleic acid sequence inhibits expression ofa target nucleic acid sequence. A transgene/heterologous coding sequencemay contain regulatory sequences operably linked to thetransgene/heterologous coding sequence (e.g., a promoter). In someembodiments, a polynucleotide sequence of interest is a transgene.However, in other embodiments, a polynucleotide sequence of interest isan endogenous nucleic acid sequence, wherein additional genomic copiesof the endogenous nucleic acid sequence are desired, or a nucleic acidsequence that is in the antisense orientation with respect to thesequence of a target nucleic acid molecule in the host organism.

As used herein, the term a transgenic “event” is produced bytransformation of plant cells with heterologous DNA, i.e., a nucleicacid construct that includes a transgene/heterologous coding sequence ofinterest, regeneration of a population of plants resulting from theinsertion of the transgene/heterologous coding sequence into the genomeof the plant, and selection of a particular plant characterized byinsertion into a particular genome location. The term “event” refers tothe original transformant and progeny of the transformant that includethe heterologous DNA. The term “event” also refers to progeny producedby a sexual outcross between the transformant and another variety thatincludes the genomic/transgene DNA. Even after repeated back-crossing toa recurrent parent, the inserted transgene/heterologous coding sequenceDNA and flanking genomic DNA (genomic/transgene DNA) from thetransformed parent is present in the progeny of the cross at the samechromosomal location. The term “event” also refers to DNA from theoriginal transformant and progeny thereof comprising the inserted DNAand flanking genomic sequence immediately adjacent to the inserted DNAthat would be expected to be transferred to a progeny that receivesinserted DNA including the transgene/heterologous coding sequence ofinterest as the result of a sexual cross of one parental line thatincludes the inserted DNA (e.g., the original transformant and progenyresulting from selfing) and a parental line that does not contain theinserted DNA.

As used herein, the terms “Polymerase Chain Reaction” or “PCR” define aprocedure or technique in which minute amounts of nucleic acid, RNAand/or DNA, are amplified as described in U.S. Pat. No. 4,683,195 issuedJul. 28, 1987. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers may coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (StocktonPress, N Y, 1989).

As used herein, the term “primer” refers to an oligonucleotide capableof acting as a point of initiation of synthesis along a complementarystrand when conditions are suitable for synthesis of a primer extensionproduct. The synthesizing conditions include the presence of fourdifferent deoxyribonucleotide triphosphates and at least onepolymerization-inducing agent such as reverse transcriptase or DNApolymerase. These are present in a suitable buffer, which may includeconstituents which are co-factors or which affect conditions such as pHand the like at various suitable temperatures. A primer is preferably asingle strand sequence, such that amplification efficiency is optimized,but double stranded sequences can be utilized.

As used herein, the term “probe” refers to an oligonucleotide thathybridizes to a target sequence. In the TaqMan® or TaqMan®-style assayprocedure, the probe hybridizes to a portion of the target situatedbetween the annealing site of the two primers. A probe includes abouteight nucleotides, about ten nucleotides, about fifteen nucleotides,about twenty nucleotides, about thirty nucleotides, about fortynucleotides, or about fifty nucleotides. In some embodiments, a probeincludes from about eight nucleotides to about fifteen nucleotides. Aprobe can further include a detectable label, e.g., a fluorophore(Texas-Red®, Fluorescein isothiocyanate, etc.). The detectable label canbe covalently attached directly to the probe oligonucleotide, e.g.,located at the probe's 5′ end or at the probe's 3′ end. A probeincluding a fluorophore may also further include a quencher, e.g., BlackHole Quencher™, Iowa Black™, etc.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence. Type-2 restrictionenzymes recognize and cleave DNA at the same site, and include but arenot limited to XbaI, BamHI, HindIII, EcoRI, XhoI, SalI, KpnI, AvaI, PstIand SmaI.

As used herein, the term “vector” is used interchangeably with the terms“construct”, “cloning vector” and “expression vector” and means thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. A “non-viral vector” is intended to mean any vector that doesnot comprise a virus or retrovirus. In some embodiments a “vector” is asequence of DNA comprising at least one origin of DNA replication and atleast one selectable marker gene. Examples include, but are not limitedto, a plasmid, cosmid, bacteriophage, bacterial artificial chromosome(BAC), or virus that carries exogenous DNA into a cell. A vector canalso include one or more genes, antisense molecules, and/or selectablemarker genes and other genetic elements known in the art. A vector maytransduce, transform, or infect a cell, thereby causing the cell toexpress the nucleic acid molecules and/or proteins encoded by thevector.

The term “plasmid” defines a circular strand of nucleic acid capable ofautosomal replication in either a prokaryotic or a eukaryotic host cell.The term includes nucleic acid which may be either DNA or RNA and may besingle- or double-stranded. The plasmid of the definition may alsoinclude the sequences which correspond to a bacterial origin ofreplication.

As used herein, the term “selectable marker gene” as used herein definesa gene or other expression cassette which encodes a protein whichfacilitates identification of cells into which the selectable markergene is inserted. For example a “selectable marker gene” encompassesreporter genes as well as genes used in plant transformation to, forexample, protect plant cells from a selective agent or provideresistance/tolerance to a selective agent. In one embodiment only thosecells or plants that receive a functional selectable marker are capableof dividing or growing under conditions having a selective agent. Thephrase “marker-positive” refers to plants that have been transformed toinclude a selectable marker gene.

As used herein, the term “detectable marker” refers to a label capableof detection, such as, for example, a radioisotope, fluorescentcompound, bioluminescent compound, a chemiluminescent compound, metalchelator, or enzyme. Examples of detectable markers include, but are notlimited to, the following: fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In anembodiment, a detectable marker can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, the terms “cassette”, “expression cassette” and “geneexpression cassette” refer to a segment of DNA that can be inserted intoa nucleic acid or polynucleotide at specific restriction sites or byhomologous recombination. As used herein the segment of DNA comprises apolynucleotide that encodes a polypeptide of interest, and the cassetteand restriction sites are designed to ensure insertion of the cassettein the proper reading frame for transcription and translation. In anembodiment, an expression cassette can include a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.In an embodiment, a gene expression cassette may also include elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

As used herein a “linker” or “spacer” is a bond, molecule or group ofmolecules that binds two separate entities to one another. Linkers andspacers may provide for optimal spacing of the two entities or mayfurther supply a labile linkage that allows the two entities to beseparated from each other. Labile linkages include photocleavablegroups, acid-labile moieties, base-labile moieties and enzyme-cleavablegroups. The terms “polylinker” or “multiple cloning site” as used hereindefines a cluster of three or more Type-2 restriction enzyme siteslocated within 10 nucleotides of one another on a nucleic acid sequence.In other instances the term “polylinker” as used herein refers to astretch of nucleotides that are targeted for joining two sequences viaany known seamless cloning method (i.e., Gibson Assembly®, NEBuilderHiFiDNA Assembly®, Golden Gate Assembly, BioBrick® Assembly, etc.).Constructs comprising a polylinker are utilized for the insertion and/orexcision of nucleic acid sequences such as the coding region of a gene.

As used herein, the term “control” refers to a sample used in ananalytical procedure for comparison purposes. A control can be“positive” or “negative”. For example, where the purpose of ananalytical procedure is to detect a differentially expressed transcriptor polypeptide in cells or tissue, it is generally preferable to includea positive control, such as a sample from a known plant exhibiting thedesired expression, and a negative control, such as a sample from aknown plant lacking the desired expression.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. A class of plant that canbe used in the present invention is generally as broad as the class ofhigher and lower plants amenable to mutagenesis including angiosperms(monocotyledonous and dicotyledonous plants), gymnosperms, ferns andmulticellular algae. Thus, “plant” includes dicot and monocot plants.The term “plant parts” include any part(s) of a plant, including, forexample and without limitation: seed (including mature seed and immatureseed); a plant cutting; a plant cell; a plant cell culture; a plantorgan (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots,stems, and explants). A plant tissue or plant organ may be a seed,protoplast, callus, or any other group of plant cells that is organizedinto a structural or functional unit. A plant cell or tissue culture maybe capable of regenerating a plant having the physiological andmorphological characteristics of the plant from which the cell or tissuewas obtained, and of regenerating a plant having substantially the samegenotype as the plant. In contrast, some plant cells are not capable ofbeing regenerated to produce plants. Regenerable cells in a plant cellor tissue culture may be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks, or stalks.

Plant parts include harvestable parts and parts useful for propagationof progeny plants. Plant parts useful for propagation include, forexample and without limitation: seed; fruit; a cutting; a seedling; atuber; and a rootstock. A harvestable part of a plant may be any usefulpart of a plant, including, for example and without limitation: flower;pollen; seedling; tuber; leaf, stem; fruit; seed; and root.

A plant cell is the structural and physiological unit of the plant,comprising a protoplast and a cell wall. A plant cell may be in the formof an isolated single cell, or an aggregate of cells (e.g., a friablecallus and a cultured cell), and may be part of a higher organized unit(e.g., a plant tissue, plant organ, and plant). Thus, a plant cell maybe a protoplast, a gamete producing cell, or a cell or collection ofcells that can regenerate into a whole plant. As such, a seed, whichcomprises multiple plant cells and is capable of regenerating into awhole plant, is considered a “plant cell” in embodiments herein.

As used herein, the term “small RNA” refers to several classes ofnon-coding ribonucleic acid (ncRNA). The term small RNA describes theshort chains of ncRNA produced in bacterial cells, animals, plants, andfungi. These short chains of ncRNA may be produced naturally within thecell or may be produced by the introduction of an exogenous sequencethat expresses the short chain or ncRNA. The small RNA sequences do notdirectly code for a protein, and differ in function from other RNA inthat small RNA sequences are only transcribed and not translated. Thesmall RNA sequences are involved in other cellular functions, includinggene expression and modification. Small RNA molecules are usually madeup of about 20 to 30 nucleotides. The small RNA sequences may be derivedfrom longer precursors. The precursors form structures that fold back oneach other in self-complementary regions; they are then processed by thenuclease Dicer in animals or DCL1 in plants.

Many types of small RNA exist either naturally or produced artificially,including microRNAs (miRNAs), short interfering RNAs (siRNAs), antisenseRNA, short hairpin RNA (shRNA), and small nucleolar RNAs (snoRNAs).Certain types of small RNA, such as microRNA and siRNA, are important ingene silencing and RNA interference (RNAi). Gene silencing is a processof genetic regulation in which a gene that would normally be expressedis “turned off” by an intracellular element, in this case, the smallRNA. The protein that would normally be formed by this geneticinformation is not formed due to interference, and the information codedin the gene is blocked from expression.

As used herein, the term “small RNA” encompasses RNA molecules describedin the literature as “tiny RNA” (Storz, (2002) Science 296:1260-3;Illangasekare et al., (1999) RNA 5:1482-1489); prokaryotic “small RNA”(sRNA) (Wassarman et al., (1999) Trends Microbiol. 7:37-45); eukaryotic“noncoding RNA (ncRNA)”; “micro-RNA (miRNA)”; “small non-mRNA (snmRNA)”;“functional RNA (fRNA)”; “transfer RNA (tRNA)”; “catalytic RNA” [e.g.,ribozymes, including self-acylating ribozymes (Illangaskare et al.,(1999) RNA 5:1482-1489); “small nucleolar RNAs (snoRNAs),” “tmRNA”(a.k.a. “10S RNA,” Muto et al., (1998) Trends Biochem Sci. 23:25-29; andGillet et al., (2001) Mol Microbiol. 42:879-885); RNAi moleculesincluding without limitation “small interfering RNA (siRNA),”“endoribonuclease-prepared siRNA (e-siRNA),” “short hairpin RNA(shRNA),” and “small temporally regulated RNA (stRNA),” “diced siRNA(d-siRNA),” and aptamers, oligonucleotides and other synthetic nucleicacids that comprise at least one uracil base.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this disclosure belongs.Definitions of common terms in molecular biology can be found in, forexample: Lewin, Genes V, Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers(ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

III. GmTEFs1 Gene Regulatory Elements and Nucleic Acids Comprising theSame

Provided are methods and compositions for using a promoter from aGlycine max Glyma19g07240 (Elongation factor-1 alpha) gene to expressnon-GmTEFs1 transgenes in plant. In an embodiment, a promoter can be theGmTEFs1 gene promoter of SEQ ID NO:2.

In an embodiment, a polynucleotide is provided comprising a promoter,wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:2. Inan embodiment, a promoter is a GmTEFs1 gene promoter comprising apolynucleotide of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.8%, or 100% identity to the polynucleotide ofSEQ ID NO:2. In an embodiment, an isolated polynucleotide is providedcomprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ IDNO:2. In an embodiment, a nucleic acid vector is provided comprising aGmTEFs1 promoter of SEQ ID NO:2. In an embodiment, a polynucleotide isprovided comprising a GmTEFs1 promoter that is operably linked to apolylinker. In an embodiment, a gene expression cassette is providedcomprising a GmTEFs1 promoter that is operably linked to a non-GmTEFs1transgene. In an embodiment, a nucleic acid vector is providedcomprising a GmTEFs1 promoter that is operably linked to a non-GmTEFs1transgene. In one embodiment, the promoter consists of SEQ ID NO:2. Inan illustrative embodiment, a nucleic acid vector comprises a GmTEFs1promoter that is operably linked to a transgene, wherein thetransgene/heterologous coding sequence can be an insecticidal resistancetransgene, an herbicide tolerance transgene, a nitrogen use efficiencytransgene, a water use efficiency transgene, a nutritional qualitytransgene, a DNA binding transgene, a small RNA transgene, selectablemarker transgene, or combinations thereof.

In an embodiment, a nucleic acid vector comprises a gene expressioncassette as disclosed herein. In an embodiment, a vector can be aplasmid, a cosmid, a bacterial artificial chromosome (BAC), abacteriophage, a virus, or an excised polynucleotide fragment for use indirect transformation or gene targeting such as a donor DNA.

Transgene expression may also be regulated by a 5′ UTR region locateddownstream of the promoter sequence. Both a promoter and a 5′ UTR canregulate transgene/heterologous coding sequence expression. While apromoter is necessary to drive transcription, the presence of a 5′ UTRcan increase expression levels resulting in mRNA transcript fortranslation and protein synthesis. A 5′ UTR gene region aids stableexpression of a transgene. In a further embodiment an 5′ UTR is operablylinked to a GmTEFs1 promoter. In an embodiment, a 5′ UTR can be theGmTEFs1 5′ UTR of SEQ ID NO:3.

In an embodiment, a polynucleotide is provided comprising a 5′ UTR,wherein the 5′ UTR is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:3. Inan embodiment, a 5′ UTR is a GmTEFs1 5′ UTR comprising a polynucleotideof at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ ID NO:3. Inan embodiment, an isolated polynucleotide is provided comprising atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identity to the polynucleotide of SEQ ID NO:3. In anembodiment, a nucleic acid vector is provided comprising GmTEFs1 5′ UTRof SEQ ID NO:3. In an embodiment, a polynucleotide is providedcomprising a GmTEFs1 5′ UTR that is operably linked to a polylinker. Inan embodiment, a gene expression cassette is provided comprising aGmTEFs1 5′ UTR that is operably linked to a non-GmTEFs1 transgene. In anembodiment, a nucleic acid vector is provided comprising a GmTEFs1 5′UTR that is operably linked to a non-GmTEFs1 transgene. In oneembodiment, the 5′ UTR consists of SEQ ID NO:3. In an illustrativeembodiment, a nucleic acid vector comprises a GmTEFs1 5′ UTR that isoperably linked to a transgene, wherein the transgene/heterologouscoding sequence can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a small RNA transgene, selectable marker transgene,or combinations thereof.

Transgene expression may also be regulated by an intron region locateddownstream of the promoter sequence. Both a promoter and an intron canregulate transgene/heterologous coding sequence expression. While apromoter is necessary to drive transcription, the presence of an introncan increase expression levels resulting in mRNA transcript fortranslation and protein synthesis. An intron gene region aids stableexpression of a transgene. In a further embodiment an intron is operablylinked to a GmTEFs1 promoter. In an embodiment, a intron can be theGmTEFs1 5′ UTR of SEQ ID NO:28.

In an embodiment, a polynucleotide is provided comprising a intron,wherein intron is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:28. In anembodiment, a intron is a GmTEFs1 intron comprising a polynucleotide ofat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ ID NO:28. Inan embodiment, an isolated polynucleotide is provided comprising atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identity to the polynucleotide of SEQ ID NO:28. In anembodiment, a nucleic acid vector is provided comprising GmTEFs1 intronof SEQ ID NO:28. In an embodiment, a polynucleotide is providedcomprising a GmTEFs1 intron that is operably linked to a polylinker. Inan embodiment, a gene expression cassette is provided comprising aGmTEFs1 intron that is operably linked to a non-GmTEFs1 transgene. In anembodiment, a nucleic acid vector is provided comprising a GmTEFs1intron that is operably linked to a non-GmTEFs1 transgene. In oneembodiment, the intron consists of SEQ ID NO:28. In an illustrativeembodiment, a nucleic acid vector comprises a GmTEFs1 intron that isoperably linked to a transgene, wherein the transgene/heterologouscoding sequence can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a small RNA transgene, selectable marker transgene,or combinations thereof.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene expression cassette wherein therecombinant gene expression cassette comprises a GmTEFs1 promoteroperably linked to a polylinker sequence, a non-GmTEFs1 gene ornon-GmTEFs1 transgene or combination thereof. In one embodiment therecombinant gene cassette comprises a GmTEFs1 promoter operably linkedto a non-GmTEFs1 gene or transgene. In one embodiment the recombinantgene cassette comprises a GmTEFs1 promoter as disclosed herein isoperably linked to a polylinker sequence. The polylinker is operablylinked to the GmTEFs1 promoter in a manner such that insertion of acoding sequence into one of the restriction sites of the polylinker willoperably link the coding sequence allowing for expression of the codingsequence when the vector is transformed or transfected into a host cell.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of a GmTEFs1 promoter and anon-GmTEFs1 gene. In an embodiment, the GmTEFs1 promoter of SEQ ID NO: 2is operably linked to the 5′ end of the non-GmTEFs1 gene or transgene.In a further embodiment the GmTEFs1 promoter sequence comprises SEQ IDNO:2 or a sequence that has 80, 85, 90, 95, 99 or 100% sequence identitywith SEQ ID NO:2. In accordance with one embodiment a nucleic acidvector is provided comprising a gene cassette that consists of a GmTEFs1promoter, a non-GmTEFs1 gene, wherein the GmTEFs1 promoter is operablylinked to the 5′ end of the non-GmTEFs1 gene, and the GmTEFs1 promotersequence comprises SEQ ID NO:2 or a sequence that has 80, 85, 90, 95, 99or 100% sequence identity with SEQ ID NO: 2. In a further embodiment theGmTEFs1 promoter sequence consists of SEQ ID NO: 2, or a 371 bp sequencethat has 80, 85, 90, 95, or 99% sequence identity with SEQ ID NO: 2.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene expression cassette wherein therecombinant gene expression cassette comprises a GmTEFs1 5′ UTR operablylinked to a polylinker sequence, a non-GmTEFs1 gene or transgene orcombination thereof. In one embodiment the recombinant gene cassettecomprises a GmTEFs1 5′ UTR operably linked to a non-GmTEFs1 gene ortransgene. In one embodiment the recombinant gene cassette comprises aGmTEFs1 5′ UTR as disclosed herein is operably linked to a polylinkersequence. The polylinker is operably linked to the GmTEFs1 5′ UTR in amanner such that insertion of a coding sequence into one of therestriction sites of the polylinker will operably link the codingsequence allowing for expression of the coding sequence when the vectoris transformed or transfected into a host cell.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of a GmTEFs1 5′ UTR and anon-GmTEFs1 gene. In an embodiment, the GmTEFs1 5′ UTR of SEQ ID NO:3 isoperably linked to the 5′ end of the non-GmTEFs1 gene or transgene. In afurther embodiment the GmTEFs1 5′ UTR sequence comprises SEQ ID NO:3 ora sequence that has 80, 85, 90, 95, 99 or 100% sequence identity withSEQ ID NO:3. In accordance with one embodiment a nucleic acid vector isprovided comprising a gene cassette that consists of a GmTEFs1 5′ UTR, anon-GmTEFs1 gene, wherein the GmTEFs1 5′ UTR is operably linked to the5′ end of the non-GmTEFs1 gene, and the GmTEFs1 gene 5′ UTR sequencecomprises SEQ ID NO:3 or a sequence that has 80, 85, 90, 95, 99 or 100%sequence identity with SEQ ID NO:3. In a further embodiment the GmTEFs1gene 5′ UTR sequence consists of SEQ ID NO:3, or a 248 bp sequence thathas 80, 85, 90, 95, or 99% sequence identity with SEQ ID NO:3.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene expression cassette wherein therecombinant gene expression cassette comprises a GmTEFs1 intron operablylinked to a polylinker sequence, a non-GmTEFs1 gene or transgene orcombination thereof. In one embodiment the recombinant gene cassettecomprises a GmTEFs1 intron operably linked to a non-GmTEFs1 gene ortransgene. In one embodiment the recombinant gene cassette comprises aGmTEFs1 intron as disclosed herein is operably linked to a polylinkersequence. The polylinker is operably linked to the GmTEFs1 intron in amanner such that insertion of a coding sequence into one of therestriction sites of the polylinker will operably link the codingsequence allowing for expression of the coding sequence when the vectoris transformed or transfected into a host cell.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of a GmTEFs1 intron and anon-GmTEFs1 gene. In an embodiment, the GmTEFs1 intron of SEQ ID NO:28is operably linked to the 5′ end of the non-GmTEFs1 gene or transgene.In a further embodiment the GmTEFs1 intron sequence comprises SEQ IDNO:28 or a sequence that has 80, 85, 90, 95, 99 or 100% sequenceidentity with SEQ ID NO:28. In accordance with one embodiment a nucleicacid vector is provided comprising a gene cassette that consists of aGmTEFs1 intron, a non-GmTEFs1 gene, wherein the GmTEFs1 intron isoperably linked to the 5′ end of the non-GmTEFs1 gene, and the GmTEFs1gene intron sequence comprises SEQ ID NO:28 or a sequence that has 80,85, 90, 95, 99 or 100% sequence identity with SEQ ID NO:28. In a furtherembodiment the GmTEFs1 gene intron sequence consists of SEQ ID NO:28, ora 895 bp sequence that has 80, 85, 90, 95, or 99% sequence identity withSEQ ID NO:28.

A GmTEFs1 promoter may also comprise one or more additional sequenceelements. In some embodiments, a GmTEFs1 promoter may comprise an exon(e.g., a leader or signal peptide such as a chloroplast transit peptideor ER retention signal). For example and without limitation, a GmTEFs1promoter may encode an exon incorporated into the GmTEFs1 promoter as afurther embodiment.

Further provided are methods and compositions for using a 3′ UTR from aGlycine max Glyma19g07240 (Elongation factor-1 alpha) gene to terminatethe expression of non-GmTEFs1 transgenes in a plant. In an embodiment, a3′ UTR terminator can be the GmTEFs1 3′ UTR of SEQ ID NO:4.

In an embodiment, a polynucleotide is provided comprising a 3′ UTR,wherein the 3′ UTR is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:4. Inan embodiment, a 3′ UTR is a GmTEFs1 3′ UTR comprising a polynucleotideof at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ ID NO:4. Inan embodiment, an isolated polynucleotide is provided comprising atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identity to the polynucleotide of SEQ ID NO:4. In anembodiment, a nucleic acid vector is provided comprising a GmTEFs1 3′UTR of SEQ ID NO:4. In an embodiment, a polynucleotide is providedcomprising a GmTEFs1 3′ UTR that is operably linked to a polylinker. Inan embodiment, a gene expression cassette is provided comprising aGmTEFs1 3′ UTR that is operably linked to a non-GmTEFs1 transgene. In anembodiment, a nucleic acid vector is provided comprising a GmTEFs1 3′UTR that is operably linked to a non-GmTEFs1 transgene. In oneembodiment, the 3′ UTR consists of SEQ ID NO: 4. In an illustrativeembodiment, a nucleic acid vector comprises a GmTEFs1 gene 3′ UTR thatis operably linked to a transgene, wherein the transgene/heterologouscoding sequence can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a small RNA transgene, selectable marker transgene,or combinations thereof.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene expression cassette wherein therecombinant gene expression cassette comprises a GmTEFs1 3′UTR operablylinked to a polylinker sequence, a non-GmTEFs1 gene ortransgene/heterologous coding sequence or combination thereof. In oneembodiment the recombinant gene cassette comprises a GmTEFs1 3′UTRoperably linked to a non-GmTEFs1 gene or transgene. In one embodimentthe recombinant gene cassette comprises a GmTEFs1 3′UTR as disclosedherein is operably linked to a polylinker sequence. The polylinker isoperably linked to the GmTEFs1 3′UTR in a manner such that insertion ofa coding sequence into one of the restriction sites of the polylinkerwill operably link the coding sequence allowing for expression of thecoding sequence when the vector is transformed or transfected into ahost cell.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of a GmTEFs1 3′UTR and anon-GmTEFs1 gene. In an embodiment, the GmTEFs1 3′UTR of SEQ ID NO:4 isoperably linked to the 3′ end of the non-GmTEFs1 gene or transgene. In afurther embodiment the GmTEFs1 3′UTR sequence comprises SEQ ID NO:4 or asequence that has 80, 85, 90, 95, 99 or 100% sequence identity with SEQID NO:4. In accordance with one embodiment a nucleic acid vector isprovided comprising a gene cassette that consists of a GmTEFs1 3′UTR, anon-GmTEFs1 gene, wherein the GmTEFs1 3′UTR is operably linked to the 3′end of the non-GmTEFs1 gene, and the GmTEFs1 3′UTR sequence comprisesSEQ ID NO:4 or a sequence that has 80, 85, 90, 95, 99 or 100% sequenceidentity with SEQ ID NO:4. In a further embodiment the GmTEFs1 3′UTRsequence consists of SEQ ID NO:4, or a 739 bp sequence that has 80, 85,90, 95, or 99% sequence identity with SEQ ID NO:4.

Further provided are methods and compositions for using a terminatorfrom a Glycine max Glyma19g07240 (Elongation factor-1 alpha) gene toterminate the expression of non-GmTEFs1 transgenes in a plant. In anembodiment, a terminator can be the GmTEFs1 terminator of SEQ ID NO:5.

In an embodiment, a polynucleotide is provided comprising a terminator,wherein the terminator is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:5.In an embodiment, a terminator is a GmTEFs1 terminator comprising apolynucleotide of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.8%, or 100% identity to the polynucleotide ofSEQ ID NO:5. In an embodiment, an isolated polynucleotide is providedcomprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ IDNO:5. In an embodiment, a nucleic acid vector is provided comprising aGmTEFs1 terminator of SEQ ID NO:5. In an embodiment, a polynucleotide isprovided comprising a GmTEFs1 terminator that is operably linked to apolylinker. In an embodiment, a gene expression cassette is providedcomprising a GmTEFs1 terminator that is operably linked to a non-GmTEFs1transgene. In an embodiment, a nucleic acid vector is providedcomprising a GmTEFs1 terminator that is operably linked to a non-GmTEFs1transgene. In one embodiment, the terminator consists of SEQ ID NO: 5.In an illustrative embodiment, a nucleic acid vector comprises a GmTEFs1terminator that is operably linked to a transgene, wherein thetransgene/heterologous coding sequence can be an insecticidal resistancetransgene, an herbicide tolerance transgene, a nitrogen use efficiencytransgene, a water use efficiency transgene, a nutritional qualitytransgene, a DNA binding transgene, a small RNA transgene, selectablemarker transgene, or combinations thereof.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene expression cassette wherein therecombinant gene expression cassette comprises a GmTEFs1 terminatoroperably linked to a polylinker sequence, a non-GmTEFs1 gene ortransgene or combination thereof. In one embodiment the recombinant genecassette comprises a GmTEFs1 terminator operably linked to a non-GmTEFs1gene or transgene. In one embodiment the recombinant gene cassettecomprises a GmTEFs1 terminator as disclosed herein is operably linked toa polylinker sequence. The polylinker is operably linked to the GmTEFs1terminator in a manner such that insertion of a coding sequence into oneof the restriction sites of the polylinker will operably link the codingsequence allowing for expression of the coding sequence when the vectoris transformed or transfected into a host cell.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of a GmTEFs1 terminator and anon-GmTEFs1 gene. In an embodiment, the GmTEFs1 terminator of SEQ IDNO:5 is operably linked to the 3′ end of the non-GmTEFs1 gene ortransgene. In a further embodiment the GmTEFs1 terminator sequencecomprises SEQ ID NO:5 or a sequence that has 80, 85, 90, 95, 99 or 100%sequence identity with SEQ ID NO:5. In accordance with one embodiment anucleic acid vector is provided comprising a gene cassette that consistsof a GmTEFs1 terminator, a non-GmTEFs1 gene, wherein the GmTEFs1terminator is operably linked to the 3′ end of the non-GmTEFs1 gene, andthe GmTEFs1 terminator sequence comprises SEQ ID NO:5 or a sequence thathas 80, 85, 90, 95, 99 or 100% sequence identity with SEQ ID NO:5. In afurther embodiment the GmTEFs1 terminator sequence consists of SEQ IDNO:5, or a 897 bp sequence that has 80, 85, 90, 95, or 99% sequenceidentity with SEQ ID NO:5.

In one embodiment a nucleic acid construct is provided comprising aGmTEFs1 promoter and a non-GmTEFs1 gene and optionally one or more ofthe following elements:

a) a 5′ untranslated region;

b) an intron; and

c) a 3′ untranslated region,

wherein,

the GmTEFs1 promoter consists of SEQ ID NO:2 or a sequence having 95%sequence identity with SEQ ID NO:2;

the GmTEFs1 5′UTR consists of a known 5′UTR, SEQ ID NO:3 or a sequencehaving 95% sequence identity with SEQ ID NO:3; and

the 3′ UTR consists of a known 3′ UTR, SEQ ID NO:4 or a sequence having95% sequence identity with SEQ ID NO:4; further wherein said GmTEFs1promoter is operably linked to said transgene/heterologous codingsequence and each optional element, when present, is also operablylinked to both the promoter and the transgene. In a further embodiment atransgenic cell is provided comprising the nucleic acid constructdisclosed immediately above. In one embodiment the transgenic cell is aplant cell, and in a further embodiment a plant is provided wherein theplant comprises said transgenic cells.

In one embodiment a nucleic acid construct is provided comprising aGmTEFs1 promoter and a non-GmTEFs1 gene and optionally one or more ofthe following elements:

a) a 5′ untranslated region;

b) an intron; and

c) a 3′ terminator region,

wherein,

the GmTEFs1 promoter consists of SEQ ID NO:2 or a sequence having 95%sequence identity with SEQ ID NO:2;

the GmTEFs1 5′UTR consists of a known 5′UTR, SEQ ID NO:3 or a sequencehaving 95% sequence identity with SEQ ID NO:3; and

the 3′ terminator consists of a known 3′ terminator, SEQ ID NO:5 or asequence having 95% sequence identity with SEQ ID NO:5; further whereinsaid GmTEFs1 promoter is operably linked to said transgene/heterologouscoding sequence and each optional element, when present, is alsooperably linked to both the promoter and the transgene. In a furtherembodiment a transgenic cell is provided comprising the nucleic acidconstruct disclosed immediately above. In one embodiment the transgeniccell is a plant cell, and in a further embodiment a plant is providedwherein the plant comprises said transgenic cells.

Another aspect of the subject disclosure comprises a functional variantwhich differs in one or more nucleotides from those of the nucleotidesequences comprising the regulatory element, provided herein. Such avariant is produced as the result of one or more modifications (e.g.,deletion, rearrangement, or insertion) of the nucleotide sequencescomprising the sequence described herein. For example, fragments andvariants of the GmTEFs1 promoter sequence of SEQ ID NO: 2 may be used ina DNA construct or in a gene expression cassette to drive expression ofa heterologous coding sequence. As used herein, the term “fragment”refers to a portion of the nucleic acid sequence. Fragments of GmTEFs1promoter sequence of SEQ ID NO: 2 may retain the biological activity ofinitiating transcription, more particularly driving transcription in atissue constitutively expressed manner. Alternatively, fragments of anucleotide sequence which are useful as hybridization probes may notnecessarily retain biological activity. Fragments of a nucleotidesequence for the promoter region of the GmTEFs1 promoter sequence of SEQID NO:2 may range from at least about 20 nucleotides, about 50nucleotides, about 100 nucleotides, up to the full-length nucleotidesequence of the invention for the promoter region of the gene.

A biologically active portion of a GmTEFs1 promoter sequence of SEQ IDNO:2 can be prepared by isolating a portion of the GmTEFs1 promotersequence of SEQ ID NO:2, and assessing the promoter activity of theportion. Nucleic acid molecules that are fragments of an GmTEFs1promoter nucleotide sequence comprise at least about 16, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1550, 1600, 1650, or 1700nucleotides, or up to the number of nucleotides present in a full-lengthGmTEFs1 promoter sequence disclosed herein.

Variant nucleotide sequences also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. With sucha procedure, GmTEFs1 promoter nucleotide sequences of SEQ ID NO:2 can bemanipulated to create a new GmTEFs1 promoter. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA i:10747-10751; Stemmer (1994) Nature 570:389-391; Crameri et al. (1997)Nature Biotech. 75:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA£4:4504-4509; Crameri et al. (1998) Nature 527:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the subject disclosure can be used toisolate corresponding sequences from other organisms, particularly otherplants, more particularly other monocots. In this manner, methods suchas PCR, hybridization, and the like can be used to identify suchsequences based on their sequence homology to the sequences set forthherein. Sequences isolated based on their sequence identity to theentire GmTEFs1 promoter sequence set forth herein or to fragmentsthereof are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from genomic DNAextracted from any plant of interest. Methods for designing PCR primersand PCR cloning are generally known in the art and are disclosed inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafterSambrook. See also Innis et al., eds. (1990) PCR Protocols: A Guide toMethods and Applications (Academic Press, New York); Innis and Gelfand,eds. (1995) PCR Strategies (Academic Press, New York); and Innis andGelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).Known methods of PCR include, but are not limited to, methods usingpaired primers, nested primers, single specific primers, degenerateprimers, gene-specific primers, vector-specific primers,partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments from a chosen organism. The hybridization probes may belabeled with a detectable group such as P³² or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the GmTEFs1 promotersequence of the invention. Methods for preparation of probes forhybridization and for construction of genomic libraries are generallyknown in the art and are disclosed in Sambrook. For example, the entireGmTEFs1 promoter sequence disclosed herein, or one or more portionsthereof, may be used as a probe capable of specifically hybridizing tocorresponding GmTEFs1 promoter sequences and messenger RNAs. To achievespecific hybridization under a variety of conditions, such probesinclude sequences that are unique among GmTEFs1 promoter sequence andare at least about 10 nucleotides in length or at least about 20nucleotides in length. Such probes may be used to amplify correspondingGmTEFs1 promoter sequence from a chosen plant by PCR. This technique maybe used to isolate additional coding sequences from a desired organism,or as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook).

In accordance with one embodiment the nucleic acid vector furthercomprises a sequence encoding a selectable maker. In accordance with oneembodiment the recombinant gene cassette is operably linked to anAgrobacterium T-DNA border. In accordance with one embodiment therecombinant gene cassette further comprises a first and second T-DNAborder, wherein the first T-DNA border is operably linked to one end ofa gene construct, and the second T-DNA border is operably linked to theother end of a gene construct. The first and second Agrobacterium T-DNAborders can be independently selected from T-DNA border sequencesoriginating from bacterial strains selected from the group consisting ofa nopaline synthesizing Agrobacterium T-DNA border, an ocotopinesynthesizing Agrobacterium T-DNA border, a mannopine synthesizingAgrobacterium T-DNA border, a succinamopine synthesizing AgrobacteriumT-DNA border, or any combination thereof. In one embodiment anAgrobacterium strain selected from the group consisting of a nopalinesynthesizing strain, a mannopine synthesizing strain, a succinamopinesynthesizing strain, or an octopine synthesizing strain is provided,wherein said strain comprises a plasmid wherein the plasmid comprises atransgene/heterologous coding sequence operably linked to a sequenceselected from SEQ ID NO:2 or a sequence having 80, 85, 90, 95, or 99%sequence identity with SEQ ID NO:2. In another embodiment, the first andsecond Agrobacterium T-DNA borders can be independently selected fromT-DNA border sequences originating from bacterial strains selected fromthe group consisting of a nopaline synthesizing Agrobacterium T-DNAborder, an ocotopine synthesizing Agrobacterium T-DNA border, amannopine synthesizing Agrobacterium T-DNA border, a succinamopinesynthesizing Agrobacterium T-DNA border, or any combination thereof. Inan embodiment an Agrobacterium strain selected from the group consistingof a nopaline synthesizing strain, a mannopine synthesizing strain, asuccinamopine synthesizing strain, or an octopine synthesizing strain isprovided, wherein said strain comprises a plasmid wherein the plasmidcomprises a transgene/heterologous coding sequence operably linked to asequence selected from SEQ ID NO:3 or a sequence having 80, 85, 90, 95,or 99% sequence identity with SEQ ID NO:3. In one embodiment anAgrobacterium strain selected from the group consisting of a nopalinesynthesizing strain, a mannopine synthesizing strain, a succinamopinesynthesizing strain, or an octopine synthesizing strain is provided,wherein said strain comprises a plasmid wherein the plasmid comprises atransgene/heterologous coding sequence operably linked to a sequenceselected from SEQ ID NO:4 or a sequence having 80, 85, 90, 95, or 99%sequence identity with SEQ ID NO:4. In one embodiment an Agrobacteriumstrain selected from the group consisting of a nopaline synthesizingstrain, a mannopine synthesizing strain, a succinamopine synthesizingstrain, or an octopine synthesizing strain is provided, wherein saidstrain comprises a plasmid wherein the plasmid comprises atransgene/heterologous coding sequence operably linked to a sequenceselected from SEQ ID NO:5 or a sequence having 80, 85, 90, 95, or 99%sequence identity with SEQ ID NO:5.

Transgenes of interest that are suitable for use in the presentdisclosed constructs include, but are not limited to, coding sequencesthat confer (1) resistance to pests or disease, (2) tolerance toherbicides, (3) value added agronomic traits, such as; yieldimprovement, nitrogen use efficiency, water use efficiency, andnutritional quality, (4) binding of a protein to DNA in a site specificmanner, (5) expression of small RNA, and (6) selectable markers. Inaccordance with one embodiment, the transgene/heterologous codingsequence encodes a selectable marker or a gene product conferringinsecticidal resistance, herbicide tolerance, small RNA expression,nitrogen use efficiency, water use efficiency, or nutritional quality.

1. Insect Resistance

Various insect resistance genes can be operably linked to the GmTEFs1promoter comprising SEQ ID NO: 2, or a sequence that has 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 2. In addition, the insectresistance genes can be operably linked to the GmTEFs1 5′ UTR comprisingSEQ ID NO:3, or a sequence that has 80, 85, 90, 95 or 99% sequenceidentity with SEQ ID NO:3. The insect resistance genes can be operablylinked to the GmTEFs1 intron comprising SEQ ID NO:28, or a sequence thathas 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO:28. Likewise,the insect resistance genes can be operably linked to the GmTEFs1 3′ UTRcomprising SEQ ID NO:4, or a sequence that has 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO: 4. Furthermore, the insect resistancegenes can be operably linked to the GmTEFs1 terminator comprising SEQ IDNO:5, or a sequence that has 80, 85, 90, 95 or 99% sequence identitywith SEQ ID NO: 5. The operably linked sequences can then beincorporated into a chosen vector to allow for identification andselection of transformed plants (“transformants”). Exemplary insectresistance coding sequences are known in the art. As embodiments ofinsect resistance coding sequences that can be operably linked to theregulatory elements of the subject disclosure, the following traits areprovided. Coding sequences that provide exemplary Lepidopteran insectresistance include: cry1A; cry1A.105; cry1Ab; cry1Ab (truncated);cry1Ab-Ac (fusion protein); cry1Ac (marketed as Widestrike®); cry1C;cry1F (marketed as Widestrike®); cryFa2; cry2Ab2; cry2Ae; cry9C;mocryiF; pinI (protease inhibitor protein); vip3A(a); and vip3Aa20.Coding sequences that provide exemplary Coleopteran insect resistanceinclude: cry34Ab1 (marketed as Herculex®); cry35Ab1 (marketed asHerculex®); cry3A; cry3Bb; dvsnf7; and mcry3A. Coding sequences thatprovide exemplary multi-insect resistance include ecry31.Ab. The abovelist of insect resistance genes is not meant to be limiting. Any insectresistance genes are encompassed by the present disclosure.

2. Herbicide Tolerance

Various herbicide tolerance genes can be operably linked to the GmTEFs1promoter comprising SEQ ID NO: 2, or a sequence that has 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 2. In addition, the insectresistance genes can be operably linked to the GmTEFs1 5′ UTR comprisingSEQ ID NO:3, or a sequence that has 80, 85, 90, 95 or 99% sequenceidentity with SEQ ID NO:3. The insect resistance genes can be operablylinked to the GmTEFs1 intron comprising SEQ ID NO:28, or a sequence thathas 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO:28. Likewise,the insect resistance genes can be operably linked to the GmTEFs1 3′ UTRcomprising SEQ ID NO:4, or a sequence that has 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO: 4. Furthermore, the insect resistancegenes can be operably linked to the GmTEFs1 terminator comprising SEQ IDNO:5, or a sequence that has 80, 85, 90, 95 or 99% sequence identitywith SEQ ID NO: 5. The operably linked sequences can then beincorporated into a chosen vector to allow for identification andselection of transformed plants (“transformants”). Exemplary herbicidetolerance coding sequences are known in the art. As embodiments ofherbicide tolerance coding sequences that can be operably linked to theregulatory elements of the subject disclosure, the following traits areprovided. The glyphosate herbicide contains a mode of action byinhibiting the EPSPS enzyme (5-enolpyruvylshikimate-3-phosphatesynthase). This enzyme is involved in the biosynthesis of aromatic aminoacids that are essential for growth and development of plants. Variousenzymatic mechanisms are known in the art that can be utilized toinhibit this enzyme. The genes that encode such enzymes can be operablylinked to the gene regulatory elements of the subject disclosure. In anembodiment, selectable marker genes include, but are not limited togenes encoding glyphosate resistance genes include: mutant EPSPS genessuch as 2mEPSPS genes, cp4 EPSPS genes, mEPSPS genes, dgt-28 genes; aroAgenes; and glyphosate degradation genes such as glyphosate acetyltransferase genes (gat) and glyphosate oxidase genes (gox). These traitsare currently marketed as Gly-Tol™, Optimum® GAT®, Agrisure® GT andRoundup Ready®. Resistance genes for glufosinate and/or bialaphoscompounds include dsm-2, bar and pat genes. The bar andpat traits arecurrently marketed as LibertyLink®. Also included are tolerance genesthat provide resistance to 2,4-D such as aad-1 genes (it should be notedthat aad-1 genes have further activity on arloxyphenoxypropionateherbicides) and aad-12 genes (it should be noted that aad-12 genes havefurther activity on pyidyloxyacetate synthetic auxins). These traits aremarketed as Enlist® crop protection technology. Resistance genes for ALSinhibitors (sulfonylureas, imidazolinones, triazolopyrimidines,pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) areknown in the art. These resistance genes most commonly result from pointmutations to the ALS encoding gene sequence. Other ALS inhibitorresistance genes include hra genes, the csrl-2 genes, Sr-HrA genes, andsurB genes. Some of the traits are marketed under the tradenameClearfield®. Herbicides that inhibit HPPD include the pyrazolones suchas pyrazoxyfen, benzofenap, and topramezone; triketones such asmesotrione, sulcotrione, tembotrione, benzobicyclon; and diketonitrilessuch as isoxaflutole. These exemplary HPPD herbicides can be toleratedby known traits. Examples of HPPD inhibitors include hppdPF_W336 genes(for resistance to isoxaflutole) and avhppd-03 genes (for resistance tomeostrione). An example of oxynil herbicide tolerant traits include thebxn gene, which has been showed to impart resistance to theherbicide/antibiotic bromoxynil. Resistance genes for dicamba includethe dicamba monooxygenase gene (dmo) as disclosed in International PCTPublication No. WO 2008/105890. Resistance genes for PPO or PROTOXinhibitor type herbicides (e.g., acifluorfen, butafenacil, flupropazil,pentoxazone, carfentrazone, fluazolate, pyraflufen, aclonifen,azafenidin, flumioxazin, flumiclorac, bifenox, oxyfluorfen, lactofen,fomesafen, fluoroglycofen, and sulfentrazone) are known in the art.Exemplary genes conferring resistance to PPO include over expression ofa wild-type Arabidopsis thaliana PPO enzyme (Lermontova I and Grimm B,(2000) Overexpression of plastidic protoporphyrinogen IX oxidase leadsto resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol122:75-83), the B. subtilis PPO gene (Li, X. and Nicholl D. 2005.Development of PPO inhibitor-resistant cultures and crops. Pest Manag.Sci. 61:277-285 and Choi K W, Han O, Lee H J, Yun Y C, Moon Y H, Kim MK, Kuk Y I, Han S U and Guh J O, (1998) Generation of resistance to thediphenyl ether herbicide, oxyfluorfen, via expression of the Bacillussubtilis protoporphyrinogen oxidase gene in transgenic tobacco plants.Biosci Biotechnol Biochem 62:558-560.) Resistance genes for pyridinoxyor phenoxy proprionic acids and cyclohexones include the ACCaseinhibitor-encoding genes (e.g., Accl-S1, Accl-S2 and Accl-S3). Exemplarygenes conferring resistance to cyclohexanediones and/oraryloxyphenoxypropanoic acid include haloxyfop, diclofop, fenoxyprop,fluazifop, and quizalofop. Finally, herbicides can inhibitphotosynthesis, including triazine or benzonitrile are providedtolerance by psbA genes (tolerance to triazine), 1s+ genes (tolerance totriazine), and nitrilase genes (tolerance to benzonitrile). The abovelist of herbicide tolerance genes is not meant to be limiting. Anyherbicide tolerance genes are encompassed by the present disclosure.

3. Agronomic Traits

Various agronomic trait genes can be operably linked to the GmTEFs1promoter comprising SEQ ID NO: 2, or a sequence that has 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 2. In addition, the insectresistance genes can be operably linked to the GmTEFs1 5′ UTR comprisingSEQ ID NO:3, or a sequence that has 80, 85, 90, 95 or 99% sequenceidentity with SEQ ID NO:3. The insect resistance genes can be operablylinked to the GmTEFs1 intron comprising SEQ ID NO:28, or a sequence thathas 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO:28. Likewise,the insect resistance genes can be operably linked to the GmTEFs1 3′ UTRcomprising SEQ ID NO:4, or a sequence that has 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO: 4. Furthermore, the insect resistancegenes can be operably linked to the GmTEFs1 terminator comprising SEQ IDNO:5, or a sequence that has 80, 85, 90, 95 or 99% sequence identitywith SEQ ID NO: 5. The operably linked sequences can then beincorporated into a chosen vector to allow for identification andselection of transformed plants (“transformants”). Exemplary agronomictrait coding sequences are known in the art. As embodiments of agronomictrait coding sequences that can be operably linked to the regulatoryelements of the subject disclosure, the following traits are provided.Delayed fruit softening as provided by the pg genes inhibit theproduction of polygalacturonase enzyme responsible for the breakdown ofpectin molecules in the cell wall, and thus causes delayed softening ofthe fruit. Further, delayed fruit ripening/senescence of acc genes actto suppress the normal expression of the native acc synthase gene,resulting in reduced ethylene production and delayed fruit ripening.Whereas, the accd genes metabolize the precursor of the fruit ripeninghormone ethylene, resulting in delayed fruit ripening. Alternatively,the sam-k genes cause delayed ripening by reducing S-adenosylmethionine(SAM), a substrate for ethylene production. Drought stress tolerancephenotypes as provided by cspB genes maintain normal cellular functionsunder water stress conditions by preserving RNA stability andtranslation. Another example includes the EcBetA genes that catalyze theproduction of the osmoprotectant compound glycine betaine conferringtolerance to water stress. In addition, the RmBetA genes catalyze theproduction of the osmoprotectant compound glycine betaine conferringtolerance to water stress. Photosynthesis and yield enhancement isprovided with the bbx32 gene that expresses a protein that interactswith one or more endogenous transcription factors to regulate theplant's day/night physiological processes. Ethanol production can beincrease by expression of the amy797E genes that encode a thermostablealpha-amylase enzyme that enhances bioethanol production by increasingthe thermostability of amylase used in degrading starch. Finally,modified amino acid compositions can result by the expression of thecordapA genes that encode a dihydrodipicolinate synthase enzyme thatincreases the production of amino acid lysine. The above list ofagronomic trait coding sequences is not meant to be limiting. Anyagronomic trait coding sequence is encompassed by the presentdisclosure.

4. DNA Binding Proteins

Various DNA binding transgene/heterologous coding sequencegenes/heterologous coding sequences can be operably linked to theGmTEFs1 promoter comprising SEQ ID NO: 2, or a sequence that has 80, 85,90, 95 or 99% sequence identity with SEQ ID NO: 2. In addition, theinsect resistance genes can be operably linked to the GmTEFs1 5′ UTRcomprising SEQ ID NO:3, or a sequence that has 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO:3. The insect resistance genes can beoperably linked to the GmTEFs1 intron comprising SEQ ID NO:28, or asequence that has 80, 85, 90, 95 or 99% sequence identity with SEQ IDNO:28. Likewise, the insect resistance genes can be operably linked tothe GmTEFs1 3′ UTR comprising SEQ ID NO:4, or a sequence that has 80,85, 90, 95 or 99% sequence identity with SEQ ID NO: 4. Furthermore, theinsect resistance genes can be operably linked to the GmTEFs1 terminatorcomprising SEQ ID NO:5, or a sequence that has 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO: 5. The operably linked sequences canthen be incorporated into a chosen vector to allow for identificationand selectable of transformed plants (“transformants”). Exemplary DNAbinding protein coding sequences are known in the art. As embodiments ofDNA binding protein coding sequences that can be operably linked to theregulatory elements of the subject disclosure, the following types ofDNA binding proteins can include; Zinc Fingers, TALENS, CRISPRS, andmeganucleases. The above list of DNA binding protein coding sequences isnot meant to be limiting. Any DNA binding protein coding sequences isencompassed by the present disclosure.

5. Small RNA

Various small RNA sequences can be operably linked to the GmTEFs1promoter comprising SEQ ID NO: 2, or a sequence that has 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 2. In addition, the insectresistance genes can be operably linked to the GmTEFs1 5′ UTR comprisingSEQ ID NO:3, or a sequence that has 80, 85, 90, 95 or 99% sequenceidentity with SEQ ID NO:3. The insect resistance genes can be operablylinked to the GmTEFs1 intron comprising SEQ ID NO:28, or a sequence thathas 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO:28. Likewise,the insect resistance genes can be operably linked to the GmTEFs1 3′ UTRcomprising SEQ ID NO:4, or a sequence that has 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO: 4. Furthermore, the insect resistancegenes can be operably linked to the GmTEFs1 terminator comprising SEQ IDNO:5, or a sequence that has 80, 85, 90, 95 or 99% sequence identitywith SEQ ID NO: 5. The operably linked sequences can then beincorporated into a chosen vector to allow for identification andselection of transformed plants (“transformants”). Exemplary small RNAtraits are known in the art. As embodiments of small RNA codingsequences that can be operably linked to the regulatory elements of thesubject disclosure, the following traits are provided. For example,delayed fruit ripening/senescence of the anti-efe small RNA delaysripening by suppressing the production of ethylene via silencing of theACO gene that encodes an ethylene-forming enzyme. The altered ligninproduction of ccomt small RNA reduces content of guanacyl (G) lignin byinhibition of the endogenous S-adenosyl-L-methionine: trans-caffeoyl CoA3-O-methyltransferase (CCOMT gene). Further, the Black Spot BruiseTolerance in Solanum verrucosum can be reduced by the Ppo5 small RNAwhich triggers the degradation of Ppo5 transcripts to block black spotbruise development. Also included is the dvsnf7 small RNA that inhibitsWestern Corn Rootworm with dsRNA containing a 240 bp fragment of theWestern Corn Rootworm Snf7 gene. Modified starch/carbohydrates canresult from small RNA such as the pPhL small RNA (degrades PhLtranscripts to limit the formation of reducing sugars through starchdegradation) and pR1 small RNA (degrades R1 transcripts to limit theformation of reducing sugars through starch degradation). Additional,benefits such as reduced acrylamide resulting from the asn1 small RNAthat triggers degradation of Asn1 to impair asparagine formation andreduce polyacrylamide. Finally, the non-browning phenotype of pgas pposuppression small RNA results in suppressing PPO to produce apples witha non-browning phenotype. The above list of small RNAs is not meant tobe limiting. Any small RNA encoding sequences are encompassed by thepresent disclosure.

6. Selectable Markers

Various selectable markers also described as reporter genes can beoperably linked to the GmTEFs1 promoter comprising SEQ ID NO: 2, or asequence that has 80, 85, 90, 95 or 99% sequence identity with SEQ IDNO: 2. In addition, the insect resistance genes can be operably linkedto the GmTEFs1 5′ UTR comprising SEQ ID NO:3, or a sequence that has 80,85, 90, 95 or 99% sequence identity with SEQ ID NO:3. The insectresistance genes can be operably linked to the GmTEFs1 intron comprisingSEQ ID NO:28, or a sequence that has 80, 85, 90, 95 or 99% sequenceidentity with SEQ ID NO:28. Likewise, the insect resistance genes can beoperably linked to the GmTEFs1 3′ UTR comprising SEQ ID NO:4, or asequence that has 80, 85, 90, 95 or 99% sequence identity with SEQ IDNO: 4. Furthermore, the insect resistance genes can be operably linkedto the GmTEFs1 terminator comprising SEQ ID NO:5, or a sequence that has80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 5. The operablylinked sequences can then be incorporated into a chosen vector to allowfor identification and selectable of transformed plants(“transformants”). Many methods are available to confirm expression ofselectable markers in transformed plants, including for example DNAsequencing and PCR (polymerase chain reaction), Southern blotting, RNAblotting, immunological methods for detection of a protein expressedfrom the vector. But, usually the reporter genes are observed throughvisual observation of proteins that when expressed produce a coloredproduct. Exemplary reporter genes are known in the art and encodeβ-glucuronidase (GUS), luciferase, green fluorescent protein (GFP),yellow fluorescent protein (YFP, Phi-YFP), redfluorescent protein(DsRFP, RFP, etc), β-galactosidase, and the like (See Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Press, N.Y., 2001, the content of which is incorporated herein byreference in its entirety).

Selectable marker genes are utilized for selection of transformed cellsor tissues. Selectable marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO),spectinomycin/streptinomycin resistance (AAD), and hygromycinphosphotransferase (HPT or HGR) as well as genes conferring resistanceto herbicidal compounds. Herbicide resistance genes generally code for amodified target protein insensitive to the herbicide or for an enzymethat degrades or detoxifies the herbicide in the plant before it canact. For example, resistance to glyphosate has been obtained by usinggenes coding for mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and mutantsfor EPSPS are well known, and further described below. Resistance toglufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D)have been obtained by using bacterial genes encoding PAT or DSM-2, anitrilase, an AAD-1, or an AAD-12, each of which are examples ofproteins that detoxify their respective herbicides.

In an embodiment, herbicides can inhibit the growing point or meristem,including imidazolinone or sulfonylurea, and genes forresistance/tolerance of acetohydroxyacid synthase (AHAS) andacetolactate synthase (ALS) for these herbicides are well known.Glyphosate resistance genes include mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes(via the introduction of recombinant nucleic acids and/or various formsof in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosateacetyl transferase (GAT) genes, respectively). Resistance genes forother phosphono compounds include bar and pat genes from Streptomycesspecies, including Streptomyces hygroscopicus and Streptomycesviridichromogenes, and pyridinoxy or phenoxy proprionic acids andcyclohexones (ACCase inhibitor-encoding genes). Exemplary genesconferring resistance to cyclohexanediones and/oraryloxyphenoxypropanoic acid (including haloxyfop, diclofop, fenoxyprop,fluazifop, quizalofop) include genes of acetyl coenzyme A carboxylase(ACCase); Accl-S1, Accl-S2 and Accl-S3. In an embodiment, herbicides caninhibit photosynthesis, including triazine (psbA and 1s+ genes) orbenzonitrile (nitrilase gene). Futhermore, such selectable markers caninclude positive selection markers such as phosphomannose isomerase(PMI) enzyme.

In an embodiment, selectable marker genes include, but are not limitedto genes encoding: 2,4-D; neomycin phosphotransferase II; cyanamidehydratase; aspartate kinase; dihydrodipicolinate synthase; tryptophandecarboxylase; dihydrodipicolinate synthase and desensitized aspartatekinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase(NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolatereductase (DHFR); phosphinothricin acetyltransferase;2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase;5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase;acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32kD photosystem II polypeptide (psbA). An embodiment also includesselectable marker genes encoding resistance to: chloramphenicol;methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; andphosphinothricin. The above list of selectable marker genes is not meantto be limiting. Any reporter or selectable marker gene are encompassedby the present disclosure.

In some embodiments the coding sequences are synthesized for optimalexpression in a plant. For example, in an embodiment, a coding sequenceof a gene has been modified by codon optimization to enhance expressionin plants. An insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, ora selectable marker transgene/heterologous coding sequence can beoptimized for expression in a particular plant species or alternativelycan be modified for optimal expression in dicotyledonous ormonocotyledonous plants. Plant preferred codons may be determined fromthe codons of highest frequency in the proteins expressed in the largestamount in the particular plant species of interest. In an embodiment, acoding sequence, gene, heterologous coding sequence ortransgene/heterologous coding sequence is designed to be expressed inplants at a higher level resulting in higher transformation efficiency.Methods for plant optimization of genes are well known. Guidanceregarding the optimization and production of synthetic DNA sequences canbe found in, for example, W2013016546, WO2011146524, WO1997013402, U.S.Pat. Nos. 6,166,302, and 5,380,831, herein incorporated by reference.

Transformation

Suitable methods for transformation of plants include any method bywhich DNA can be introduced into a cell, for example and withoutlimitation: electroporation (see, e.g., U.S. Pat. No. 5,384,253);micro-projectile bombardment (see, e.g., U.S. Pat. Nos. 5,015,580,5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865);Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos.5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301); andprotoplast transformation (see, e.g., U.S. Pat. No. 5,508,184).

A DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as agitation with silicon carbidefibers (see, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA particle bombardment (see, e.g., Klein et al.(1987) Nature 327:70-73). Alternatively, the DNA construct can beintroduced into the plant cell via nanoparticle transformation (see,e.g., US Patent Publication No. 20090104700, which is incorporatedherein by reference in its entirety).

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassava vein mosaic virus and/or tobacco mosaic virus, See, e.g.,Chung et al. (2006) Trends Plant Sci. 11(1):1-4.

Through the application of transformation techniques, cells of virtuallyany plant species may be stably transformed, and these cells may bedeveloped into transgenic plants by well-known techniques. For example,techniques that may be particularly useful in the context of cottontransformation are described in U.S. Pat. Nos. 5,846,797, 5,159,135,5,004,863, and 6,624,344; techniques for transforming Brassica plants inparticular are described, for example, in U.S. Pat. No. 5,750,871;techniques for transforming soy bean are described, for example, in U.S.Pat. No. 6,384,301; and techniques for transforming Zea mays aredescribed, for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616, andInternational PCT Publication WO 95/06722.

After effecting delivery of an exogenous nucleic acid to a recipientcell, a transformed cell is generally identified for further culturingand plant regeneration. In order to improve the ability to identifytransformants, one may desire to employ a selectable marker gene withthe transformation vector used to generate the transformant. In anillustrative embodiment, a transformed cell population can be assayed byexposing the cells to a selective agent or agents, or the cells can bescreened for the desired marker gene trait.

Cells that survive exposure to a selective agent, or cells that havebeen scored positive in a screening assay, may be cultured in media thatsupports regeneration of plants. In an embodiment, any suitable planttissue culture media may be modified by including further substances,such as growth regulators. Tissue may be maintained on a basic mediawith growth regulators until sufficient tissue is available to beginplant regeneration efforts, or following repeated rounds of manualselection, until the morphology of the tissue is suitable forregeneration (e.g., at least 2 weeks), then transferred to mediaconducive to shoot formation. Cultures are transferred periodicallyuntil sufficient shoot formation has occurred. Once shoots are formed,they are transferred to media conducive to root formation. Oncesufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

Transgenic Plants

In an embodiment, a plant, plant tissue, or plant cell comprises aGmTEFs1 promoter. In one embodiment a plant, plant tissue, or plant cellcomprises the GmTEFs1 promoter of a sequence selected from SEQ ID NO:2or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequence identitywith a sequence selected from SEQ ID NO:2. In an embodiment, a plant,plant tissue, or plant cell comprises a gene expression cassettecomprising a sequence selected from SEQ ID NO:2, or a sequence that has80%, 85%, 90%, 95% or 99.5% sequence identity with a sequence selectedfrom SEQ ID NO:2 that is operably linked to a non-GmTEFs1 gene. In anillustrative embodiment, a plant, plant tissue, or plant cell comprisesa gene expression cassette comprising a GmTEFs1 promoter that isoperably linked to a transgene or heterologous coding sequence, whereinthe transgene or heterologous coding sequence can be an insecticidalresistance transgene, an herbicide tolerance transgene, a nitrogen useefficiency transgene, a water use efficiency transgene, a nutritionalquality transgene, a DNA binding transgene, a selectable markertransgene, or combinations thereof.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises aGmTEFs1 promoter derived sequence operably linked to a transgene,wherein the GmTEFs1 promoter derived sequence comprises a sequence SEQID NO:2 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:2. In one embodiment a plant, plant tissue, orplant cell is provided wherein the plant, plant tissue, or plant cellcomprises SEQ ID NO:2, or a sequence that has 80%, 85%, 90%, 95% or99.5% sequence identity with SEQ ID NO:2 operably linked to anon-GmTEFs1 gene. In one embodiment the plant, plant tissue, or plantcell is a dicotyledonous or monocotyledonous plant or a cell or tissuederived from a dicotyledonous or monocotyledonous plant. In oneembodiment the plant is selected from the group consisting of Zea mays,wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton,sunflower, and canola. In one embodiment the plant is Zea mays. Inanother embodiment the plant is soybean (e.g., Glycine max). Inaccordance with one embodiment the plant, plant tissue, or plant cellcomprises SEQ ID NO: 2 or a sequence having 80%, 85%, 90%, 95% or 99.5%sequence identity with SEQ ID NO:2 operably linked to a non-GmTEFs1gene. In one embodiment the plant, plant tissue, or plant cell comprisesa promoter operably linked to a transgene/heterologous coding sequencewherein the promoter consists of SEQ ID NO:2 or a sequence having 80%,85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:2. In accordancewith one embodiment the gene construct comprising GmTEFs1 promotersequence operably linked to a transgene/heterologous coding sequence isincorporated into the genome of the plant, plant tissue, or plant cell.

In an embodiment, a plant, plant tissue, or plant cell comprises aGmTEFs1 5′ UTR. In one embodiment a plant, plant tissue, or plant cellcomprises the GmTEFs1 5′ UTR of a sequence selected from SEQ ID NO:3 ora sequence that has 80%, 85%, 90%, 95% or 99.5% sequence identity with asequence selected from SEQ ID NO:3. In an embodiment, a plant, planttissue, or plant cell comprises a gene expression cassette comprising asequence selected from SEQ ID NO:3, or a sequence that has 80%, 85%,90%, 95% or 99.5% sequence identity with a sequence selected from SEQ IDNO:3 that is operably linked to a non-GmTEFs1 gene. In an illustrativeembodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a GmTEFs1 5′ UTR that is operably linkedto a transgene, wherein the transgene/heterologous coding sequence canbe an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises aGmTEFs1 5′ UTR derived sequence operably linked to a transgene, whereinthe GmTEFs1 5′ UTR derived sequence comprises a sequence SEQ ID NO:3 ora sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQID NO:3. In one embodiment a plant, plant tissue, or plant cell isprovided wherein the plant, plant tissue, or plant cell comprises SEQ IDNO:3, or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:3 operably linked to a non-GmTEFs1 gene. In oneembodiment the plant, plant tissue, or plant cell is a dicotyledonous ormonocotyledonous plant or a cell or tissue derived from a dicotyledonousor monocotyledonous plant. In one embodiment the plant is selected fromthe group consisting of Zea mays, wheat, rice, sorghum, oats, rye,bananas, sugar cane, soybean, cotton, sunflower, and canola. In oneembodiment the plant is Zea mays. In another embodiment the plant issoybean (e.g., Glycine max). In accordance with one embodiment theplant, plant tissue, or plant cell comprises SEQ ID NO:3 or a sequencehaving 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:3operably linked to a non-GmTEFs1 gene. In one embodiment the plant,plant tissue, or plant cell comprises a 5′ UTR operably linked to atransgene/heterologous coding sequence wherein the 5′ UTR consists ofSEQ ID NO:3 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:3. In accordance with one embodiment the geneconstruct comprising GmTEFs1 5′ UTR sequence operably linked to atransgene/heterologous coding sequence is incorporated into the genomeof the plant, plant tissue, or plant cell.

In an embodiment, a plant, plant tissue, or plant cell comprises aGmTEFs1 intron. In one embodiment a plant, plant tissue, or plant cellcomprises the GmTEFs1 intron of a sequence selected from SEQ ID NO:28 ora sequence that has 80%, 85%, 90%, 95% or 99.5% sequence identity with asequence selected from SEQ ID NO:28. In an embodiment, a plant, planttissue, or plant cell comprises a gene expression cassette comprising asequence selected from SEQ ID NO:28, or a sequence that has 80%, 85%,90%, 95% or 99.5% sequence identity with a sequence selected from SEQ IDNO:28 that is operably linked to a non-GmTEFs1 gene. In an illustrativeembodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a GmTEFs1 intron that is operably linkedto a transgene, wherein the transgene/heterologous coding sequence canbe an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises aGmTEFs1 intron derived sequence operably linked to a transgene, whereinthe GmTEFs1 intron derived sequence comprises a sequence SEQ ID NO:28 ora sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQID NO:28. In one embodiment a plant, plant tissue, or plant cell isprovided wherein the plant, plant tissue, or plant cell comprises SEQ IDNO:28, or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:28 operably linked to a non-GmTEFs1 gene. In oneembodiment the plant, plant tissue, or plant cell is a dicotyledonous ormonocotyledonous plant or a cell or tissue derived from a dicotyledonousor monocotyledonous plant. In one embodiment the plant is selected fromthe group consisting of Zea mays, wheat, rice, sorghum, oats, rye,bananas, sugar cane, soybean, cotton, sunflower, and canola. In oneembodiment the plant is Zea mays. In another embodiment the plant issoybean (e.g., Glycine max). In accordance with one embodiment theplant, plant tissue, or plant cell comprises SEQ ID NO:28 or a sequencehaving 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:28operably linked to a non-GmTEFs1 gene. In one embodiment the plant,plant tissue, or plant cell comprises a intron operably linked to atransgene/heterologous coding sequence wherein the intron consists ofSEQ ID NO:28 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:28. In accordance with one embodiment the geneconstruct comprising GmTEFs1 intron sequence operably linked to atransgene/heterologous coding sequence is incorporated into the genomeof the plant, plant tissue, or plant cell.

In an embodiment, a plant, plant tissue, or plant cell comprises aGmTEFs1 3′ UTR. In one embodiment a plant, plant tissue, or plant cellcomprises the GmTEFs1 3′ UTR of a sequence selected from SEQ ID NO:4 ora sequence that has 80%, 85%, 90%, 95% or 99.5% sequence identity with asequence selected from SEQ ID NO:4. In an embodiment, a plant, planttissue, or plant cell comprises a gene expression cassette comprising asequence selected from SEQ ID NO:4, or a sequence that has 80%, 85%,90%, 95% or 99.5% sequence identity with a sequence selected from SEQ IDNO:4 that is operably linked to a non-GmTEFs1 gene. In an illustrativeembodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a GmTEFs1 3′ UTR that is operably linkedto a transgene, wherein the transgene/heterologous coding sequence canbe an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises aGmTEFs1 3′ UTR derived sequence operably linked to a transgene, whereinthe GmTEFs1 3′ UTR derived sequence comprises a sequence SEQ ID NO:4 ora sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQID NO:4. In one embodiment a plant, plant tissue, or plant cell isprovided wherein the plant, plant tissue, or plant cell comprises SEQ IDNO:4, or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:4 operably linked to a non-GmTEFs1 gene. In oneembodiment the plant, plant tissue, or plant cell is a dicotyledonous ormonocotyledonous plant or a cell or tissue derived from a dicotyledonousor monocotyledonous plant. In one embodiment the plant is selected fromthe group consisting of Zea mays, wheat, rice, sorghum, oats, rye,bananas, sugar cane, soybean, cotton, sunflower, and canola. In oneembodiment the plant is Zea mays. In another embodiment the plant issoybean (e.g., Glycine max). In accordance with one embodiment theplant, plant tissue, or plant cell comprises SEQ ID NO:4 or a sequencehaving 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:4operably linked to a non-GmTEFs1 gene. In one embodiment the plant,plant tissue, or plant cell comprises a 3′ UTR operably linked to atransgene/heterologous coding sequence wherein the 3′ UTR consists ofSEQ ID NO:4 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:4. In accordance with one embodiment the geneconstruct comprising GmTEFs1 gene 3′ UTR sequence operably linked to atransgene/heterologous coding sequence is incorporated into the genomeof the plant, plant tissue, or plant cell.

In an embodiment, a plant, plant tissue, or plant cell comprises aGmTEFs1 terminator. In one embodiment a plant, plant tissue, or plantcell comprises the GmTEFs1 terminator of a sequence selected from SEQ IDNO:5 or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with a sequence selected from SEQ ID NO:5. In an embodiment, aplant, plant tissue, or plant cell comprises a gene expression cassettecomprising a sequence selected from SEQ ID NO:5, or a sequence that has80%, 85%, 90%, 95% or 99.5% sequence identity with a sequence selectedfrom SEQ ID NO:5 that is operably linked to a non-GmTEFs1 gene. In anillustrative embodiment, a plant, plant tissue, or plant cell comprisesa gene expression cassette comprising a GmTEFs1 terminator that isoperably linked to a transgene, wherein the transgene/heterologouscoding sequence can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises aGmTEFs1 terminator derived sequence operably linked to a transgene,wherein the GmTEFs1 terminator derived sequence comprises a sequence SEQID NO:5 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:5. In one embodiment a plant, plant tissue, orplant cell is provided wherein the plant, plant tissue, or plant cellcomprises SEQ ID NO:5, or a sequence that has 80%, 85%, 90%, 95% or99.5% sequence identity with SEQ ID NO:5 operably linked to anon-GmTEFs1 gene. In one embodiment the plant, plant tissue, or plantcell is a dicotyledonous or monocotyledonous plant or a cell or tissuederived from a dicotyledonous or monocotyledonous plant. In oneembodiment the plant is selected from the group consisting of Zea mays,wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton,sunflower, and canola. In one embodiment the plant is Zea mays. Inanother embodiment the plant is soybean (e.g., Glycine max). Inaccordance with one embodiment the plant, plant tissue, or plant cellcomprises SEQ ID NO:5 or a sequence having 80%, 85%, 90%, 95% or 99.5%sequence identity with SEQ ID NO:5 operably linked to a non-GmTEFs1gene. In one embodiment the plant, plant tissue, or plant cell comprisesa terminator operably linked to a transgene/heterologous coding sequencewherein the terminator consists of SEQ ID NO:5 or a sequence having 80%,85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:5. In accordancewith one embodiment the gene construct comprising GmTEFs1 geneterminator sequence operably linked to a transgene/heterologous codingsequence is incorporated into the genome of the plant, plant tissue, orplant cell.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be a dicotyledonous plant. Thedicotyledonous plant, plant tissue, or plant cell can be, but notlimited to alfalfa, rapeseed, canola, Indian mustard, Ethiopian mustard,soybean, sunflower, cotton, beans, broccoli, cabbage, cauliflower,celery, cucumber, eggplant, lettuce; melon, pea, pepper, peanut, potato,pumpkin, radish, spinach, sugarbeet, sunflower, tobacco, tomato, andwatermelon.

One of skill in the art will recognize that after the exogenous sequenceis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above, wherein the seed has the transgene/heterologous codingsequence or gene construct containing the gene regulatory elements ofthe subject disclosure. The present disclosure further encompasses theprogeny, clones, cell lines or cells of the transgenic plants describedabove wherein said progeny, clone, cell line or cell has thetransgene/heterologous coding sequence or gene construct containing thegene regulatory elements of the subject disclosure.

The present disclosure also encompasses the cultivation of transgenicplants described above, wherein the transgenic plant has thetransgene/heterologous coding sequence or gene construct containing thegene regulatory elements of the subject disclosure. Accordingly, suchtransgenic plants may be engineered to, inter alia, have one or moredesired traits or transgenic events containing the gene regulatoryelements of the subject disclosure, by being transformed with nucleicacid molecules according to the invention, and may be cropped orcultivated by any method known to those of skill in the art.

Method of Expressing a Transgene

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a GmTEFs1 promoter operably linked to at least onetransgene/heterologous coding sequence or a polylinker sequence. In anembodiment the GmTEFs1 promoter consists of a sequence selected from SEQID NO:2 or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with a sequence selected from SEQ ID NO:2. In an embodiment, amethod of expressing at least one transgene/heterologous coding sequencein a plant comprising growing a plant comprising a GmTEFs1 promoteroperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene/heterologous coding sequence in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a GmTEFs1 promoter operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a gene expression cassette comprising a GmTEFs1promoter operably linked to at least one transgene. In one embodimentthe GmTEFs1 promoter consists of a sequence selected from SEQ ID NO:2 ora sequence that has 80%, 85%, 90%, 95% or 99.5% sequence identity with asequence selected from SEQ ID NO:2. In an embodiment, a method ofexpressing at least one transgene/heterologous coding sequence in aplant comprises growing a plant comprising a gene expression cassettecomprising a GmTEFs1 promoter operably linked to at least one transgene.In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a gene expression cassette comprising a GmTEFs1promoter operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene/heterologous coding sequencein a plant tissue or plant cell comprises culturing a plant tissue orplant cell comprising a gene expression cassette containing a GmTEFs1promoter operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene/heterologous coding sequencein a plant tissue or plant cell comprises culturing a plant tissue orplant cell comprising a gene expression cassette, a GmTEFs1 promoteroperably linked to at least one transgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a GmTEFs1 5′ UTR operably linked to at least onetransgene/heterologous coding sequence or a polylinker sequence. In anembodiment the GmTEFs1 5′ UTR consists of a sequence selected from SEQID NO:3 or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with a sequence selected from SEQ ID NO:3. In an embodiment, amethod of expressing at least one transgene/heterologous coding sequencein a plant comprising growing a plant comprising a GmTEFs1 5′ UTRoperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene/heterologous coding sequence in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a GmTEFs1 5′ UTR operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a gene expression cassette comprising a GmTEFs1 5′ UTRoperably linked to at least one transgene. In one embodiment the GmTEFs15′ UTR consists of a sequence selected from SEQ ID NO:3 or a sequencethat has 80%, 85%, 90%, 95% or 99.5% sequence identity with a sequenceselected from SEQ ID NO:3. In an embodiment, a method of expressing atleast one transgene/heterologous coding sequence in a plant comprisesgrowing a plant comprising a gene expression cassette comprising aGmTEFs1 5′ UTR operably linked to at least one transgene. In anembodiment, a method of expressing at least one transgene/heterologouscoding sequence in a plant comprises growing a plant comprising a geneexpression cassette comprising a GmTEFs1 5′ UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene/heterologous coding sequence in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette containing a GmTEFs1 5′ UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene/heterologous coding sequence in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette, a GmTEFs1 5′ UTR operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a GmTEFs1 intron operably linked to at least onetransgene/heterologous coding sequence or a polylinker sequence. In anembodiment the GmTEFs1 intron consists of a sequence selected from SEQID NO:28 or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with a sequence selected from SEQ ID NO:28. In an embodiment, amethod of expressing at least one transgene/heterologous coding sequencein a plant comprising growing a plant comprising a GmTEFs1 intronoperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene/heterologous coding sequence in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a GmTEFs1 intron operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a gene expression cassette comprising a GmTEFs1 intronoperably linked to at least one transgene. In one embodiment the GmTEFs1intron consists of a sequence selected from SEQ ID NO:28 or a sequencethat has 80%, 85%, 90%, 95% or 99.5% sequence identity with a sequenceselected from SEQ ID NO:28. In an embodiment, a method of expressing atleast one transgene/heterologous coding sequence in a plant comprisesgrowing a plant comprising a gene expression cassette comprising aGmTEFs1 intron operably linked to at least one transgene. In anembodiment, a method of expressing at least one transgene/heterologouscoding sequence in a plant comprises growing a plant comprising a geneexpression cassette comprising a GmTEFs1 intron operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene/heterologous coding sequence in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette containing a GmTEFs1 intron operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene/heterologous coding sequence in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette, a GmTEFs1 intron operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a GmTEFs1 3′ UTR operably linked to at least onetransgene/heterologous coding sequence or a polylinker sequence. In anembodiment the GmTEFs1 3′ UTR consists of a sequence selected from SEQID NO:4 or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with a sequence selected from SEQ ID NO:4. In an embodiment, amethod of expressing at least one transgene/heterologous coding sequencein a plant comprising growing a plant comprising a GmTEFs1 3′ UTRoperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene/heterologous coding sequence in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a GmTEFs1 3′ UTR operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a gene expression cassette comprising a GmTEFs1 3′ UTRoperably linked to at least one transgene. In one embodiment the GmTEFs13′ UTR consists of a sequence selected from SEQ ID NO:4 or a sequencethat has 80%, 85%, 90%, 95% or 99.5% sequence identity with a sequenceselected from SEQ ID NO:4. In an embodiment, a method of expressing atleast one transgene/heterologous coding sequence in a plant comprisesgrowing a plant comprising a gene expression cassette comprising aGmTEFs1 3′ UTR operably linked to at least one transgene. In anembodiment, a method of expressing at least one transgene/heterologouscoding sequence in a plant comprises growing a plant comprising a geneexpression cassette comprising a GmTEFs1 3′ UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene/heterologous coding sequence in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette containing a GmTEFs1 3′ UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene/heterologous coding sequence in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette, a GmTEFs1 3′ UTR operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a GmTEFs1 terminator operably linked to at least onetransgene/heterologous coding sequence or a polylinker sequence. In anembodiment the GmTEFs1 terminator consists of a sequence selected fromSEQ ID NO:5 or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequenceidentity with a sequence selected from SEQ ID NO:5. In an embodiment, amethod of expressing at least one transgene/heterologous coding sequencein a plant comprising growing a plant comprising a GmTEFs1 terminatoroperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene/heterologous coding sequence in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a GmTEFs1 terminator operably linked to at least onetransgene.

In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a gene expression cassette comprising a GmTEFs1terminator operably linked to at least one transgene. In one embodimentthe GmTEFs1 terminator consists of a sequence selected from SEQ ID NO:5or a sequence that has 80%, 85%, 90%, 95% or 99.5% sequence identitywith a sequence selected from SEQ ID NO:5. In an embodiment, a method ofexpressing at least one transgene/heterologous coding sequence in aplant comprises growing a plant comprising a gene expression cassettecomprising a GmTEFs1 terminator operably linked to at least onetransgene. In an embodiment, a method of expressing at least onetransgene/heterologous coding sequence in a plant comprises growing aplant comprising a gene expression cassette comprising a GmTEFs1terminator operably linked to at least one transgene. In an embodiment,a method of expressing at least one transgene/heterologous codingsequence in a plant tissue or plant cell comprises culturing a planttissue or plant cell comprising a gene expression cassette containing aGmTEFs1 terminator operably linked to at least one transgene. In anembodiment, a method of expressing at least one transgene/heterologouscoding sequence in a plant tissue or plant cell comprises culturing aplant tissue or plant cell comprising a gene expression cassette, aGmTEFs1 terminator operably linked to at least one transgene.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. The examples should not be construed tolimit the disclosure to the particular features or embodimentsexemplified.

EXAMPLES Example 1: Identification of Regulatory Elements from SoybeanGenomic Sequences

The expression profiles of total mRNA expression for 25 soybean tissues(var. Williams 82) were obtained via Next Generation Sequencing (NGS)and were used to identify candidate soybean genes for sourcingregulatory elements. The tissues included were collected from youngseedlings (expanded cotyledons, roots, and hypocotyls), V5 (leaves andstems), and R5 (leaves, flowers, different stages of seed and poddevelopment) soybean plants. Soybean endogenous genes that exhibited thedesired expression profile were identified as potential candidates forsourcing regulatory sequences.

One of the genes with the desired expression pattern was Glyma19g07240that was expressed ubiquitously. This gene was identified as GmTEFs1gene encoding translation Elongation factor-1 alpha(UniProtKB-O04299_SOYBN) (Apweiler, Rolf, et al. “UniProt: the universalprotein knowledgebase.” Nucleic acids research 32.suppl_1 (2004):D115-D119; available at http://www.uniprot.org/), thus this gene wasdescribed as “GmTEFs1”. Regulatory sequences from the GmTEFs1 gene wereisolated and characterized for the ability to drive transgeneexpression. The promoter of the GmTEFs1 is provided herein as SEQ IDNO:2.

The regulatory sequences of the Glyma19g07240 gene (GmTEFs1) weredefined as ˜1.4 kb sequence upstream of ATG of the Glyma19g07240 genefor the promoter and 5′ untranslated leader (UTR), and ˜0.6 kbdownstream of the Glyma19g07240 gene stop codon for the 3′ UTR andterminator. To further refine the regulatory sequences additionalanalyses of the regulatory elements were completed. Putative upstreamand downstream regulatory sequences were assessed for the presence oftransposable sequences, repressive DNA (methylation) and chromatin(histone-H3-lysine-4-dimethylation, commonly abbreviated as H3K4me2)marks using methods as previously disclosed in U.S. Patent PublicationNo. 20150128309A1, herein incorporated by reference in its entirety. TheGlyma19g07240 gene DNA sequences containing the repressive DNA andchromatin marks were excluded from the sourced upstream and downstreamregulatory sequence. Long stretches (100 bp or more) of AT-richsequences (>75% AT rich) within the 5′ and 3′ sequences were alsoavoided as means to reduce difficulties with de novo synthesis of theDNA fragments.

The resulting GmTEFs1 upstream regulatory sequence contained both apromoter (SEQ ID NO:2) and 5′ UTR (SEQ ID NO:3) that contains an 895 bpintron (SEQ ID NO:28). The downstream sequences encompassed a 3′ UTR(SEQ ID NO:4) and a terminator (SEQ ID NO:4) of the GmTEFs1 gene. Theterminator sequences extended for ˜100-200 bp beyond the last knownpoly-adenylation site. Two guanine base pair were deleted from thecandidate GmTEFs1 terminator (SEQ ID NO:5) to remove undesirablerestriction sites.

Sequences of the sourced from soybean genome the GmTEFs (Glyma19g07240)gene promoter/5′UTR and terminator are provided herein.

Example 2: Cloning of the Regulatory Sequences from Soybean

The promoter, 5′ UTR and 3′ UTR/terminator sequences of the GmTEFs1 genewere synthesized by DNA2.0. A diagram of the synthetic fragment is shownin FIG. 1. A linker containing multiple cloning site was includedbetween the promoter/5′ UTR with the intron and the 3′ UTR/terminatorsequence.

The synthetic GmTEFs1 fragment (promoter/5′UTR and terminator) wascloned in a Gateway entry vector, and the RFP/AAD12 reporter gene (SEQID NO:10) was inserted between the 5′UTR and the terminator. Thereporter gene was the dual reporter encoding a translational fusionprotein containing the RFP and AAD12 polypeptides joined with the rigidhelical peptide linker, LAE(EAAAK)₅AAA described by Arai et al, (2001),Protein Eng, 14, 529-532 and Marqusee et al, (1987), Proc Natl Acad SciUSA, 84, 8898-8902. The resulting expression cassette (SEQ ID NO:11) wasmoved to a binary vector and labeled as pDAB122124. This binary vectoralso contained the Green Fluorescent Protein (GFP) gene driven by theArabidopsis Ubiquitin3 promoter and 5′ UTR (AtUbi3) and terminated bythe Arabidopsis Ubiquitin 3 terminator (AtUbi3). Likewise, the binaryvector contained the synthetic phosphinothricin N-acetyltransferase genefrom Streptomyces viridochromogenes (PAT) was driven by the Cassava veinmosaic virus promoter (CsVMV) and terminated by the Agrobacteriumtumefaciens Orf1 terminator (AtuOrf1). The GFP and PAT gene expressioncassettes are provided as SEQ ID NO:12.

Cloning steps for the GmAct7-2 and GmGAPC1 regulatory sequences weresimilar to those described above for GmTEFs1. The GmAct7-2 was tested inthe pDAB122133 construct and GmGAPC1 was tested in the pDAB122134construct.

Example 3: N. benthamiana Leaf Infiltrations and Transient Assays ofGmTEFs1, GmAct7-2 and GmGAPC1 Driven Expression of the RFP/AAD12Reporter

Next, N. benthamiana plants were grown in the greenhouse under a 16 hourphotoperiod, 27° C./24° C. The 20-24 day old plants were used fortransient expression assays. For this, the top 3-4 leaves wereinfiltrated using a mix of two modified Agrobacterium tumefaciensstrains. The first strain was used in all infiltrations and carried thepDAB112236 construct containing transgene that expressed the P19silencing suppressor (Voinnet et al, (1999), Proc Natl Acad Sci U.S.A.,96, 14147-14152). The second Agrobacterium strain was either theexperimental strain carrying a test construct (with the GmTEFs1,GmAct7-2, or GmGAPC1 regulatory elements), or a benchmark controlconstruct (Table 1). Two benchmark constructs that were used containedthe RFP/AAD12 reporter gene under the control of Arabidopsis thalianaUbiquitin 14 promoter::Arabidopsis thaliana Ubiquitin 14 terminator(AtUbi14/AtUbi14) and the Arabidopsis thaliana Ubiquitin 10promoter::Agrobacterium tumefaciens Orf23 (AtUbi10/AtuOrf23). The mixingratios were based on Optical Density (OD) readings. The density of allAgrobacterium cultures was adjusted to OD 2.0. After infiltration,plants were grown to a Conviron™ until the infiltrated leaves werecollected on the fifth day after infiltration. Fluorescence data for thereporter genes was collected using a Typhoon™ scanner from 25-30individual 1.5 cm leaf disks for each construct.

All samples from N. benthamiana were scanned on three channels;chlorophyll (488 nm blue laser, 670 nm BP30, 580 nm split), GFP (488 nmblue laser, 520 nm BP40, 580 nm split), and RFP (532 nm green laser, 580nm BP30). The photomultiplier voltage (PMT) setting used for N.benthamiana was 340 for chlorophyll, 340 for GFP and 360 for RFP.

Results of testing in N. benthamiana transient assay are shown inTable 1. Analysis of fluorescence produced by RFP/AAD12 reportertransgene revealed that the GmTEFs1 regulatory sequences resulted inmean RFP fluorescence (1283.3 pixels/area) that was significantly higher(p<0.0001) than mean background fluorescence (26.1 pixels/area). It wasobserved that the RFP/AAD12 fluorescence from the GmTEFs1 regulatorysequences was lower (p<0.0001) than the mean RFP/AAD12 fluorescence fromthe constructs driven by the benchmark regulatory elements of theAtUbi14AtUbi14 and the AtUbi101AtuOrf23; 7567.4 and 3084.5 pixels/area,respectively. The significantly higher than background RFP/AAD12fluorescence supported by the GmTEFs1 regulatory elements indicated thatthe GmTEFs1 regulatory sequences from Glyma19g07240 were functional andcan be used to drive expression of a heterologous transgene in N.benthamiana leaf transient assays.

In contrast, to the GmTEFs1 regulatory sequences which drovesignificantly higher than background mean RFP/AAD12 fluorescenceexpression, the GmAct2-2 and GmGAPC1 regulatory sequences containedwithin the pDAB122333 and pDAB122134 constructs, respectively, producedonly low levels of expression that was similar to the background (Table1). These results demonstrate that the de novo isolated GmAct2-2 andGmGAPC1 candidate soybean regulatory sequences were not able to driveRFP/AAD12 transgene expression. Lack of RFP/AAD12 expression in thepDAB122333 and pDAB122134 constructs was not due to poor infiltrationsbecause the second transgene within these constructs, GFP, displayedstrong fluorescence that was significantly higher than background(p<0.0001). Thus, these results show that the de novo candidateregulatory sequences from Glyma06g15520 and Glyma06g01850 were notcapable of driving heterologous reporter transgene expression.

Based on these results, constructs pDAB122333 and pDAB122134 carryingGmAct7-2 and GmGAPC1, respectively, were not pursued further. Incontrast, the pDAB122124 construct containing the GmTEFs1 regulatorysequences and exhibiting high constitutive levels of RFP/AAD12fluorescence, as compared to background of the N. benthamiana leaves,was advanced for further testing in stably transformed Arabidopsistransgenic plants.

TABLE 1 Results of assaying RFP/AAD12 fluorescence in transientlytransformed N. benthamiana leaves. RFP fluorescence GFP fluorescence(pixels/area) (pixels/area) Regulatory No. of Std Std Std Std ConstructElement Name Samples Mean Median Dev Err Mean Median Dev Err P19 onlyNone 216 26.1 24.0 14.4 1.0 34.6 33.5 14.0 1.0 (Background) pDABAtUbi14/ 261 7567.4*** 6698.4 5191.7 321.4 9770.9*** 9230.0 4609.7 285.3117559 AtUbi14 pDAB AtUbi10/ 260 3084.5*** 2760.0 1984.2 123.1 8915.9***8737.2 5404.7 335.2 117560 AtuOrf23 pDAB GmAct7-2/ 60 25.3 26.4 7.0 0.914206.1*** 12874.3 6178.5 797.6 122133 GmAct7-2 pDAB GmGAPC1/ 30 25.324.6 5.1 0.9 5295.7*** 5184.5 2085.8 380.8 122134 GmGAPC1 pDAB GmTEFs1/60 1283.3*** 1236.5 785.0 101.3 6952.7*** 6294.7 4459.6 575.7 122124GmTEFs1 Note: ***indicates RFP or GFP means that are significantlyhigher (p < 0.0001) than the mean background fluorescence. Due topresence of unequal variances, Welch t-test was used to compare mean RFPor GFP fluorescence of each construct to the corresponding fluorescencemeans of “P19 only” background control. Statistical analyses wereconducted using JMP ® statistical package.

Example 4: Agrobacterium-Mediated Transformation of Arabidopsis andMolecular Analyses of Transgenic Events

Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used to test therelative expression of RFP/AAD12 reporter under the control of theGmTEFs1 regulatory elements. A standard Arabidopsis transformationprocedure was used to produce transgenic seed by inflorescence dipmethod (Clough and Bent, 1998). The T₁ seeds were sown on selectiontrays (10.5″×21″×1″, T.O. Plastics Inc., Clearwater, Minn.). For this,200 mg of cold stratified seeds (0.1% agar+385 mg/L Liberty for 48 hoursbefore sowing) were distributed on selection trays using a modified airdriven spray apparatus to distribute 10 ml of seed suspension perselection tray. Trays were covered with humidity domes, marked with seedidentifier, and placed in a Conviron™ with an individual watering trayunder each flat. The humidifying dome was removed approximately fivedays post-sowing. The first watering of selection trays was done usingsub-irrigation with Hoagland's fertilizer at approximately 10-14 dayspost-sowing. In addition to stratification with the herbicide, plantsare sprayed with a 0.2% solution (20 μl/10 mL distilled H₂O) of Liberty™herbicide seven and nine days post-sowing. The T₁ plants resistant toLiberty™ were transplanted from selection trays into two inch pots andallowed to grow for seven to ten days before sampling for molecularanalysis.

Next, DNA was extracted from leaves using an approximately 0.5 squarecentimeter of Arabidopsis leaf that was pinched off each plant. Thesamples were collected in a 96-well DNA extraction plate. Then 200 μl ofextraction buffer was added to each well and tissue was disrupted withthree mm stainless steel beads using a Kleko™ tissue pulverizer (threeminutes on the maximum setting). After tissue maceration, DNA wasisolated using the BioSprint 96 DNA Plant Kit™.

For qPCR, transgene copy number was assayed using hydrolysis probedesigned to detect the pat and aad12 genes (Table 2). The Arabidopsisendogenous gene, AtTafiII15 (Arabidopsis Locus: AT4G31720), was used fornormalization of DNA template concentration (Table 2). The qPCR wasperformed as follows: 10 μl of Probes Master Mix™ with a finalconcentration of 0.4 μM of each primer and 0.2 μM of each probe. The PCRcycles were performed using 95° C. for 10 min, followed by 40amplification cycles (95° C. for 1 min, 60° C. for 40 sec, and 72° C.for 1 sec) and 40° C. for 1 sec. All qPCR assays were run in bi-plexformat, with pat or aad12 assays paired with assay for the endogenousgene AtTafII15. The cp scores, the point at which the florescence signalcrosses the background threshold using the advanced relativequantification algorithm, based on the ΔΔCt method, (LightCycler®software release 1.5) were used to analyze the real time PCR data. Allsamples were then calibrated to a known hemizygous plant to obtain thetransgene copy number. Up to 100 T₁ events that were identified as beingresistant to Liberty™ were screened to identify one and two copytransgene events that were used for further analyses of transgeneexpression in T₁ transgenic plants.

TABLE 2 Primers and probes used for genotyping and zygosity analyses of Arabidopsis transgenic plants Fluorophore Oligo name Oligo Sequencelabel Target gene AtTafII15 F SEQ ID NO: 13 — AtTafII15GAGGATTAGGGTTTCAACGGAG AtTafII15 R SEQ ID NO: 14 — AtTafII15GAGAATTGAGCTGAGACGAGG AtTafII SEQ ID NO: 15 HEX AtTafII15 15ProbeAGAGAAGTTTCGACGGATTTCGGGC PAT A primer SEQ ID NO: 16 — PATACAAGAGTGGATTGATGATCTAGAG AGGT PAT S primer SEQ ID NO: 17 — PATCTTTGATGCCTATGTGACACGTAAA CAGT PAT_AS probe SEQ ID NO: 18 Cy5 PATAGGGTGTTGTGGCTGGTATTGCTTA CGCT AAD12 F SEQ ID NO: 19 — AAD12CAGAGTCCATGCTCACCAAT AAD12 R SEQ ID NO: 20 — AAD12 ACGTGGCAACTTGAAATCCAAD12 Probe SEQ ID NO: 21 Cy5 (T1) or AAD12 TGGAGATGTGGTTGTGTGGGACAAFAM (T2)

Example 5: Evaluation of Genes Operably Linked to GmTEFs1 RegulatorySequences in T₁ Arabidopsis Plants

To evaluate expression of the RFP/AAD12 reporter gene driven by theGmTEFs1 promoter, GmTEFs1 5′ UTR and GmTEFs1 terminator regulatoryelements, single copy transgenic events were identified and assayed forRFP/AAD12 fluorescence using Typhoon instrument. All samples werescanned on three channels: chlorophyll (488 nm blue laser, 670 nm BP30,580 nm split), GFP (488 nm blue laser, 520 nm BP40, 580 nm split), andRFP (532 nm green laser, 580 nm BP30). The PMT setting for leaf tissuewas for chlorophyll 400, GFP 400 and RFP 420. For analyses offluorescence in leaves, fully expanded rosette leaves from low copy (1-2copies) transgenic events were harvested from each plant and scannedfrom adaxial (top) side. The “Contour Draw” function was used to outlineleaf shapes and normalized fluorescence was determined by dividingsignal volume by surface of the leaf. The results are shown in Table 3.

Analysis of the T₁ events for RFP/AAD12 fluorescence revealed that theGmTEFs1 regulatory elements supported high mean RFP/AAD12 fluorescence(804.4 pixels/area) that was statistically higher (p<0.0001) than themean background fluorescence (350.5 pixels/area) detected in the nontransgenic wild type control (Wt) (Table 3). These results show that theGmTEFs1 regulatory sequences drove higher than background expression ofthe RFP/AAD12 reporter in transgenic Arabidopsis thaliana plants. Themean RFP/AAD12 fluorescence produced by the GmTEFs1 regulatory elementswas lower than RFP/AAD12 fluorescence levels of the pDAB117559 andpDAB117560 benchmark constructs (p<0.0001, not shown). In the pDAB117559and pDAB117560 constructs the RFP/AAD12 reporter was under the controlof the following regulatory elements; Arabidopsis thaliana Ubiquitin 14promoter::Arabidopsis thaliana Ubiquitin 14 terminator, and theArabidopsis thaliana Ubiquitin 10 promoter::Agrobacterium tumefaciensOrf23 terminator, respectively. Although RFP/AAD12 supportedfluorescence was lower than that of the positive pDAB117559 andpDAB117560 controls, it was higher than background fluorescence,indicating that GmTEFs1 supports low but statistically higher thanbackground transgene expression. Based on these results the transgenicpDAB122124 events containing the GmTEFs1 regulatory sequences wereadvanced for further characterization in T₂ Arabidopsis.

TABLE 3 Results of testing expression of RFP/AAD12 reportergene/heterologous coding sequence expression in leaves of transgenic T₁Arabidopsis plants RFP fluorescence GFP fluorescence No. of Std Std StdStd Construct Regulatory Element events Mean Median Dev Err Mean MedianDev Err Wt None (Background) 57 350.5 269.3 231.4 30.7 665.2 614.1 218.028.9 pDAB 117559 AtUbi14/AtUbil4 60 1492.3*** 1537.3 495.2 63.95164.1*** 5164.7 1605.8 207.3 pDAB 117560 AtUbi10/AtuOrf23 63 1547.6***1556.1 504.5 63.6 5521.2*** 5515.4 1434.3 180.7 pDAB 122124GmTEFs1/GmTEFs1 15 804.4*** 766.7 248.9 64.3 6397.5*** 6369.5 741.0191.3 Note: ***indicates means that are different from the mean of themean fluorescence of the Wt control at p < 0.0001. Due to presence ofunequal variances, Welch t-test was used to compare mean RFP or GFPfluorescence of each construct to the corresponding fluorescence meansof Wt background control. Statistical analyses were conducted usingJMP ® statistical package.

Example 6: Expression of Genes Operably Linked to GmTEFs1 RegulatorySequences in Leaves of T₂ Arabidopsis Plants

The GmTEFs1 regulatory sequences exhibited lower, but significantlyhigher than background, expression levels as compared to the expressionlevels of the benchmark Arabidopsis thaliana Ubiquitin 14promoter::Arabidopsis thaliana Ubiquitin 14 terminator, and theArabidopsis thaliana Ubiquitin 10 promoter::Agrobacterium tumefaciensOrf23 terminator regulatory sequences in T₁ Arabidopsis (EXAMPLE 5).Selected events that contained the GmTEFs1 regulatory sequences drivingthe RFP/AAD12 reporter gene were advanced for further characterizationin T₂ Arabidopsis plants. Accordingly, five T₁ plants that expressedfrom medium to high levels of RFP/AAD12 and GFP were selected. Thesefive plants contained transgenic events of pDAB122124 and were selectedfor T₂ plant testing. From these four events, 56 plants were grown foreach event. The T₂ plants were molecularly genotyped as described inEXAMPLE 4. Based on molecular analyses, all homozygous and a comparablenumber of hemizygous plants were retained for fluorescence analysis ofthe single copy events. To simplify data interpretation for the two copytransgenic events, only hemizygous plants were retained for expressionanalyses.

The results of analyses in T₂ transgenic plants are provided in Table 4.The results for homozygous (1 copy) and hemizygous (1 and 2 copy) eventsthat contained the RFP/AAD12 transgene under the control of GmTEFs1regulatory elements exhibited RFP/AAD12 fluorescence that wassignificantly higher than the background fluorescence from nontransgenic control plants. While, the mean RFP/AAD12 fluorescenceproduced by hemizygous and homozygous for a transgene plants carryingthe GmTEFs1 regulatory elements was significantly higher than backgroundfluorescence (Table 4), this fluorescence was lower than that of thebenchmark pDAB117559 and pDAB117560 constructs (p<0.0002, not shown). Inthe pDAB117559 and pDAB117560 constructs the RFP/AAD12 reporter wasunder the control of the following regulatory elements; Arabidopsisthaliana Ubiquitin 14 promoter::Arabidopsis thaliana Ubiquitin 14terminator, and the Arabidopsis thaliana Ubiquitin 10promoter::Agrobacterium tumefaciens Orf23 terminator, respectively.These results demonstrate that the GmTEFs1 regulatory sequences supportthe robust expression of transgenes in two generations of transgenicevents.

TABLE 4 Results of testing expression of RFP/AAD12 reportergene/heterologous coding sequence expression in leaves of transgenic T₂Arabidopsis plants RFP fluorescence GFP fluorescence (pixels/area)(pixels/area) Regulatory No. of Std Std Std Std Construct ElementsZygocity plants Mean Median Dev Err Mean Median Dev Err Wt None none 151137.5 1062.9 384.0 99.2 337.0 322.8 60.3 15.6 (background) pDABAtUbi14/ hemi 26 10943.2*** 10394.5 2862.0 561.3 3352.3*** 3355.0 380.274.6 117559 AtUbi14 homo 40 17194.3*** 16208.0 6090.0 962.9 5649.3***6144.2 1509.4 238.7 pDAB AtUbi10/ hemi 25 8239.2*** 8031.3 1928.4 385.73436.3*** 3312.6 674.3 134.9 117560 AtuOrf23 homo 50 15334.3*** 15077.54080.2 577.0 6308.3*** 6152.4 1385.5 195.9 pDAB GmTEFs1/ hemi 262127.4*** 1938.9 577.1 113.2 2602.9*** 2680.0 360.6 70.7 122124 GmTEFs1homo 13 2881.2*** 2461.5 1182.2 327.9 3867.8*** 3550.6 1210.9 335.9Note: ***indicates means that are different from the mean of the meanfluorescence of the Wt control at p < 0.0001. Due to presence of unequalvariances, Welch t-test was used to compare mean RFP or GFP fluorescenceof each construct to the corresponding fluorescence means of Wtbackground control. Statistical analyses were conducted using JMP ®statistical package.

Interrogating the individual transgenic events (Table 5) revealed thatRFP/AAD12 fluorescence detected in the two single copy events(pDAB122124-033 and pDAB122124-065) had mean RFP/AAD12 fluorescence thatwas higher than the mean Wt fluorescence (Table 5). In these single copytransgenic events homozygous plants exhibited higher RFP/AAD12fluorescence than the hemizygous plants indicating that transgeneexpression in these events was copy number dependent. Three events thathad a complex transgene locus (two copies) also exhibited higher thanbackground RFP/AAD12 fluorescence, although levels of AAD12 expressionwere lower than that of the homozygous single copy events (p=0.0091, notshown), indicating a possibility that complex locus structures, withpotential DNA rearrangements, might have impacted RFP/AAD12 expression.

In summary, testing of the transgenic T₂ Arabidopsis events showed thatthe GmTEFs1 regulatory elements drive heritable expression of theRFP/AAD12 reporter gene that is higher than Wt background fluorescence.These results reaffirm that the GmTEFs1 regulatory elements areeffective in driving heritable transgene expression in stablytransformed Arabidopsis plants.

TABLE 5 Results of testing expression of RFP/AAD12 reportergene/heterologous coding sequence expression in leaves of homozygous andhemizygous plants of the individual T₂ Arabidopsis events Construct; RFPfluorescence GFP fluorescence Regulatory No. of (pixels/area)(pixels/area) Elements Event Zygocity plants Mean Median Std Dev Std ErrMean Median Std Dev Std Err Col-0 None none 15 1137.5 1062.9 384.0 99.2337.0 322.8 60.3 15.6 (background) pDAB 117559-057 hemi 5 10415.010400.7 930.2 416.0 3529.5 3519.1 159.8 71.5 117559; homo 8 17840.719158.8 3743.1 1323.4 6196.5 6607.7 1257.6 444.6 AtUbil4/ 117559-062hemi 6 9320.9 9586.9 808.9 330.2 3486.9 3472.2 161.6 66.0 AtUbil4 homo 914534.0 16156.6 3679.1 1226.4 5455.1 6345.2 1482.7 494.2 117559-246 hemi5 14037.8 13650.2 1112.2 497.4 3351.0 3316.9 488.5 218.4 homo 9 23442.525126.5 5547.1 1849.0 5724.0 5870.9 1573.9 524.6 117559-314 hemi 513987.2 13806.8 1671.9 747.7 3411.0 3489.6 465.9 208.3 homo 4 21176.820884.0 7321.8 3660.9 5296.8 5217.0 2391.4 1195.7 117559-391 hemi 57279.7 7364.9 922.8 412.7 2956.2 3020.1 350.4 156.7 homo 10 11855.012938.5 2711.7 857.5 5459.9 5750.6 1475.0 466.4 pDAB 117560-191 hemi 58889.2 9549.1 2092.5 935.8 3831.1 4015.2 859.5 384.4 117560; homo 1015330.1 14866.4 4404.6 1392.9 6352.1 6030.2 1756.8 555.6 AtUbi10/117560-254 hemi 5 9158.7 8167.0 2264.3 1012.6 3611.3 3358.9 664.3 297.1AtuOrf23 homo 10 14704.1 14959.2 4009.6 1267.9 6283.7 6343.9 1273.8402.8 117560-288 hemi 5 6506.8 6287.3 948.7 424.3 3066.4 3136.1 256.9114.9 homo 10 18550.4 18921.9 3158.6 998.8 7007.3 6673.1 1175.3 371.7117560-325 hemi 5 7716.5 6838.0 1951.7 872.8 3172.3 3092.6 668.9 299.1homo 10 14238.9 13152.0 4636.8 1466.3 6230.7 5924.0 1444.7 456.9117560-353 hemi 5 8924.7 8444.1 1354.0 605.5 3500.2 3459.6 733.6 328.1homo 10 13848.3 13274.9 2821.9 892.4 5667.9 5782.0 1125.1 355.8 pDAB122124-195 hemi 5 1956.82*** 1845.9 184.2 82.4 2776.7*** 2832.6 192.386.0 122124; (two copy GmTEFs1/ event) GmTEFs1 122124-033 hemi 62997.42*** 3050.55 366.7 149.7 2453.8*** 2420.2 470.9 192.28 homo 53760.4** 3670.8 1316.1 588.6 4041.2*** 4202.9 1114.1 498.2 122124-049hemi 5 1758.7* 1861.3 391.9 175.3 2554.1*** 2703.2 461.1 206.2 (two copyevent) 122124-065 hemi 5 1894.8* 1813.6 420.2 187.9 2627.3*** 2745.3325.9 145.7 homo 8 2331.6* 2246.7 713.2 252.2 3759.4*** 3301.1 1330.3470.3 122124-117 hemi 5 1855.3*** 1817.8 217.0 97.0 2632.2*** 2656.7322.0 144.0 (two copy event) Note: Statistical analyses of mean RFP andGFP fluorescence of individual pDAB122124 events were conductedseparately for each zygocity class. Due to the small sample sizes andpresence of unequal variances, Welch t-test was used to compare mean RFPor GFP fluorescence of each event to the mean of Wt controlfluorescence. Stars indicate mean RFP and GFP fluorescence of pDAB122124transgenic events (hemizygous or heterozygous) that is significantlyhigher than the mean fluorescence of the Wt control; ***p values thatare equal or smaller than 0.0002, **p values are smaller than 0.001 and*p values that equal or smaller than 0.01. Statistical analyses weredone using JMP ® statistical package.

Example 7: Soybean Transgenic Plants Production and Transgene CopyNumber Estimation Using Real Time TaqMan® PCR

The GmTEFs1 regulatory elements (pDAB122124) were further tested intransgenic soybean plants. Transgenic soybean plants were produced usingthe split seed transformation method described in Pareddy et al., US2014/0173774 A1, herein incorporated by reference in its entirety.Transgenic plantlets were analyzed molecularly to determine transgenecopy number. For this, leaf tissue samples from transgenic soybeanplants and non-transgenic controls were collected in 96-well collectiontubes. Tissue disruption was performed using tungsten 2 mm beads.Following tissue maceration, the genomic DNA was isolated in highthroughput format using the MagAttract Plant Kit™ (Qiagen, Hilden,Germany) on the Agilent BioCel™. The transgenic copy number of PAT wasdetermined by using a hydrolysis probe assay, analogous to TaqMan®assay, in bi-plex with a soybean internal reference gene, GMS116(GMFL1-25-J19 of Genbank Accession No: AK286292.1). The assays weredesigned using the LightCycler® Probe Design Software 2.0. Thetransgenic presence/absence of Spectinomycin resistance gene (SpecR) wasdetermined by using a hydrolysis probe assay, analogous to TaqMan®assay, in bi-plex with a soybean internal reference gene, GMS16. Thisassay was designed to detect the SpecR gene located within the backboneof the binary constructs used for transformation. Only events in whichthere was no amplification with SpecR probe were regenerated becausethis indicated that backbone fragments were not likely to be present inthe transgenic soybean genome. For amplification of all genes ofinterest (PAT, SpecR, GMS116), LightCycler® 480 Probes Master Mix™(Roche Applied Science) was prepared at a 1× final concentration in a 10μL volume multiplex reaction containing 0.4 μM of each primer and 0.2 μMof each probe (composition of primers and probes listed in TABLE 7). Atwo-step amplification reaction was performed using the LIGHTCYCLER 480System™ (Roche Applied Science), with an extension at 60° C. for 60seconds with fluorescence acquisition.

Analysis of real time PCR data was performed using LightCycler® softwarerelease 1.5 using the advanced relative quant module and was based onthe ΔΔCt method. For PAT, a sample of known single copy gDNA wasincluded in each run and was used as a single copy calibrator. Inaddition, each run, for all genes of interest, included a wild-type(Maverick) sample as a negative control.

TABLE 6 Primer and Probe Information for hydrolysis probe assay of PAT and SpecR genes located in the backbone and internal reference (GMS116). All sequences are indicated 5′-3′. OLIGO SEQUENCETYPE PAT F ACAAGAGTGGATTGATGATCTAGAGA (SEQ ID NO: 22) Primer PAT RCTTTGATGCCTATGTGACACGTAAAC (SEQ ID NO: 23) Primer PAT PR6FAM-CCAGCGTAAGCAATACCAGCCACAACACC-  Hydrolysis 3BHQ_1 (SEQ ID NO: 24) probe SpecR F CGCCGAAGTATCGACTCAACT (SEQ ID NO: 25) Primer SpecR RGCAACGTCGGTTCGAGATG (SEQ ID NO: 26) Primer SpecR PR6FAM-TCAGAGGTAGTTGGCGTCATCGAG-3BHQ_1 Hydrolysis (SEQ ID NO: 27) probe

Example 8: Evaluation of the GmTEFs1 Regulatory Sequences Expression inT₁ Soybean Plants

The expression of genes by the GmTEFs1 regulatory elements was tested insoybean transgenic plants. For this analysis stable transformation ofsoybean plants were generated as described in EXAMPLE 7. Transgenicplantlets carrying low transgene copy number (1-2 copies) from theGmTEFs1 regulatory elements construct (pDAB122124) and the controlbenchmark construct (pDAB117560) transformations were regenerated, andT₁ seeds were collected and used for planting in greenhouse. The T₁soybean plants were grown in greenhouse and genotyped for transgene asdescribed in EXAMPLE 7.

To evaluate expression of the RFP/AAD12 reporter gene driven by theGmTEFs1 promoter, GmTEFs1 5′ UTR and GmTEFs1 terminator regulatoryelements, transgenic plants were identified and assayed for RFP/AAD12fluorescence using Typhoon instrument. All samples were scanned on threechannels: chlorophyll (488 nm blue laser, 670 nm BP30, 580 nm split),GFP (488 nm blue laser, 520 nm BP40, 580 nm split), and RFP (532 nmgreen laser, 580 nm BP30). The PMT setting for leaf tissue was forchlorophyll 400, GFP 400 and RFP 420. Leaves were collected at V3soybean plants from the topmost fully expanded leaf. For scanning, threeleaf disks were cut out of each collected leaf. The fluorescence resultsof the RFP/AAD12 and GFP reporter genes are shown in Table 7. Theexpression of the reporter gene driven by the GmTEFs1 regulatorysequences was robust (1507.5 pixels/area for hemizygous and 2674.2pixels/area for homozygous plants) and highly significant abovebackground (613.1 pixels/area, p<0.0001, Table 7). The mean RFP/AAD12fluorescence specified by the hemizygous and homozygous pDAB122124plants carrying GmTEFs1 regulatory sequences was lower that of RFP/AAD12driven by the Arabidopsis thaliana Ubiquitin 10 promoter::Agrobacteriumtumefaciens Orf23 terminator (p<0.0001 for hemizygous and homozygousplans, not shown). Although expression of GmTEFs1 driven RFP/AAD12 waslower than that of the control, this expression level is sufficient forreliable transgene expression in T₁ soybean.

TABLE 7 Results of testing expression of RFP/AAD12 reportergene/heterologous coding sequence expression in leaves of soybean singlecopy T₁ transgenic events No. of RFP fluorescence GFP fluorescenceRegulatory Consensus No. of leaf disks (pixels/area) (pixels/area)Construct Elements zygocity plants scanned Mean Median Std Dev Std ErrMean Median Std Dev Std Err Wt Maverick Null 4 12 613.1 608.8 64.5 18 6235.4 238.0 11.6 3.3 pDAB AtUbi10/ Hemi 12 36 3075.6*** 3055.5 1036.4172.7 2084.2*** 2072.2 377.3 62.9 117560 AtuOrf23 Homo 11 33 4559.2***4300.7 2059.8 358.6 4085.2*** 4308.5 1094.8 190.6 pDAB GmTEFs1/ Hemi 2472 1507.5*** 1263.5 723.0 85.2 992.2*** 655.9 833.4 98.2 122124 GmTEFs1Homo 23 69 2674.2*** 2337.1 1305.3 157.1 2019.3*** 320.4 1877.9 226.1Note: ***indicates means that are different from the mean of the meanfluorescence of the Wt control at p < 0.0001. Due to presence of unequalvariances, Welch t-test was used to compare mean RFP or GFP fluorescenceof each construct to the corresponding fluorescence means of Wtbackground control. Statistical analyses were conducted using JMP ®statistical package.

Interrogating the individual soybean transgenic events (Table 8)revealed that RFP/AAD12 fluorescence was detected in all four singlecopy events that were assayed. For all transgenic events homozygousplants exhibited higher RFP/AAD12 fluorescence than the hemizygoussiblings, thereby indicating that transgene expression in these eventswas copy number dependent.

In summary, testing of the transgenic T₁ Soybean events showed that theGmTEFs1 regulatory elements drive heritable expression of the RFP/AAD12reporter gene that is higher than the Wt background in multipleindependent transgenic events. These results reaffirm that the GmTEFs1regulatory elements are effective in driving heritable transgeneexpression in stably transformed soybean plants.

TABLE 8 Results of testing expression of RFP/AAD12 reportergene/heterologous coding sequence expression in leaves of homozygous andhemizygous plants of the individual T2 soybean events No. of RFPfluorescence GFP fluorescence Regulatory Consensus No. of leaf(pixels/area) (pixels/area) Construct Elements Event zygocity plantsdisks Mean Medi an Std Dev Std Err Mean Median Std Dev Std Err MaverickNone none Null 4 12 613.1 608.8 64.5 18.6 235.4 238.0 11.6 3.3 pDABAtUbi10/ 117560-670 Hemi 6 18 2463.2*** 2562.1 805.5 189.9 1941.5***1875.1 439.1 103.5 117560 AtuOrf23 Homo 6 18 4002.2*** 3884.2 1918.3452.2 3631.2*** 3829.9 1203.8 283.7 117560-691 Hemi 6 18 3687.9***3528.3 876.8 206.7 2226.9*** 2172.5 239.0 56.3 Homo 5 15 5227.6 4562.02086.1 538.6 4630.0 4616.6 630.0 162.7 pDAB GmTEFsl/ 122124-213 Hemi 618 2450.0*** 2329.2 744.7 175.5 2128.5*** 2067.5 291.7 68.7 122124GmTEFsl Homo 6 18 2715.8*** 2760.2 441.1 104.0 3979.9*** 4009.2 524.1123.5 122124-220 Hemi 6 18 1066.0*** 1034.1 286.1 67.4 221.1*** 231.030.1 7.1 Homo 6 18 1852.7*** 1562.6 705.5 166.3 243.7*** 239.6 19.7 4.7122124-221 Hemi 6 18 1137.6*** 1210.5 251.8 59.3 265.0*** 270.9 58.713.8 Homo 6 18 2470.4*** 2088.6 1022.5 241.0 295.4*** 302.2 22.6 5.3122124-230 Hemi 6 18 1376.3*** 1263.5 416.6 98.2 1354.1*** 1262.3 340.180.2 Homo 3 15 3854.5*** 3228.0 1936.3 500.0 3865.9*** 3709.8 568.0146.7 Note: ***indicates means that are different from the mean of themean fluorescence of the Wt control at p < 0.0001. Due to presence ofunequal variances, Welch t-test was used to compare mean RFP or GFPfluorescence of each construct to the corresponding fluorescence meansof Wt background control. Statistical analyses were conducted usingJMP ® statistical package.

Example 9: Agrobacterium-Mediated Transformation of Genes OperablyLinked to the GmTEFs1 Promoter, the GmTEFs1 5′ UTR, the GmTEFs1 3′ UTRand/or the GmTEFs1 Terminator

Soybean may be transformed with genes operably linked to the GmTEFs1promoter, the GmTEFs1 5′ UTR, the GmTEFs1 3′ UTR and/or the GmTEFs1terminator by utilizing the same techniques previously described inExample #11 or Example #13 of patent application WO 2007/053482.

Cotton may be transformed with genes operably linked to the GmTEFs1promoter, the GmTEFs1 5′ UTR, the GmTEFs1 3′ UTR and/or the GmTEFs1terminator by utilizing the same techniques previously described inExamples #14 of U.S. Pat. No. 7,838,733 or Example #12 of patentapplication WO 2007/053482 (Wright et al.).

Canola may be transformed with genes operably linked to the GmTEFs1promoter, the GmTEFs5′ UTR, the GmTEFs1 3′ UTR and/or the GmTEFsterminator by utilizing the same techniques previously described inExample #26 of U.S. Pat. No. 7,838,733 or Example #22 of patentapplication WO 2007/053482 (Wright et al.).

Wheat may be transformed with genes operably linked to the GmTEFs1promoter, the GmTEFs1 5′ UTR, the GmTEFs1 3′ UTR and/or the GmTEFs1terminator by utilizing the same techniques previously described inExample #23 of patent application WO 2013/116700A1 (Lira et al.).

Rice may be transformed with genes operably linked to the GmTEFs1promoter, the GmTEFs1 5′ UTR, the GmTEFs1 3′ UTR and/or the GmTEFs1terminator by utilizing the same techniques previously described inExample #19 of patent application WO 2013/116700A1 (Lira et al.).

Example 10: Agrobacterium-Mediated Transformation of Genes OperablyLinked to the GmTEFs1 Regulatory Elements

In light of the subject disclosure, additional crops can be transformedaccording to embodiments of the subject disclosure using techniques thatare known in the art. For Agrobacterium-mediated transformation of rye,see, e.g., Popelka J C, Xu J, Altpeter F., “Generation of rye with lowtransgene copy number after biolistic gene transfer and production of(Secale cereale L.) plants instantly marker-free transgenic rye,”Transgenic Res. 2003 October; 12(5):587-96). For Agrobacterium-mediatedtransformation of sorghum, see, e.g., Zhao et al.,“Agrobacterium-mediated sorghum transformation,” Plant Mol Biol. 2000December; 44(6):789-98. For Agrobacterium-mediated transformation ofbarley, see, e.g., Tingay et al., “Agrobacterium tumefaciens-mediatedbarley transformation,” The Plant Journal, (1997) 11: 1369-1376. ForAgrobacterium-mediated transformation of wheat, see, e.g., Cheng et al.,“Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens,”Plant Physiol. 1997 November; 115(3):971-980. For Agrobacterium-mediatedtransformation of rice, see, e.g., Hiei et al., “Transformation of ricemediated by Agrobacterium tumefaciens,” Plant Mol. Biol. 1997 September;35(1-2):205-18.

The Latin names for these and other plants are given below. It should beclear that other (non-Agrobacterium) transformation techniques can beused to transform genes operably linked to GmTEFs1 promoter, the GmTEFs15′ UTR, the GmTEFs1 3′ UTR and/or the GmTEFs1 terminator, for example,into these and other plants. Examples include, but are not limited to;Maize (Zea mays), Wheat (Triticum spp.), Rice (Oryza spp. and Zizaniaspp.), Barley (Hordeum spp.), Cotton (Abroma augusta and Gossypiumspp.), Soybean (Glycine max), Sugar and table beets (Beta spp.), Sugarcane (Arenga pinnata), Tomato (Lycopersicon esculentum and other spp.,Physalis ixocarpa, Solanum incanum and other spp., and Cyphomandrabetacea), Potato (Solanum tuberosum), Sweet potato (Ipomoea batatas),Rye (Secale spp.), Peppers (Capsicum annuum, chinense, and frutescens),Lettuce (Lactuca sativa, perennis, and pulchella), Cabbage (Brassicaspp.), Celery (Apium graveolens), Eggplant (Solanum melongena), Peanut(Arachis hypogea), Sorghum (Sorghum spp.), Alfalfa (Medicago sativa),Carrot (Daucus carota), Beans (Phaseolus spp. and other genera), Oats(Avena sativa and strigosa), Peas (Pisum, Vigna, and Tetragonolobusspp.), Sunflower (Helianthus annuus), Squash (Cucurbita spp.), Cucumber(Cucumis sativa), Tobacco (Nicotiana spp.), Arabidopsis (Arabidopsisthaliana), Turfgrass (Lolium, Agrostis, Poa, Cynodon, and other genera),Clover (Trifolium), Vetch (Vicia). Transformation of such plants, withgenes operably linked to the GmTEFs1 promoter, the GmTEFs1 5′ UTR, theGmTEFs1 3′ UTR and/or the GmTEFs1 terminator, for example, iscontemplated in embodiments of the subject disclosure.

Use of the GmTEFs1 promoter, the GmTEFs1 5′ UTR, the GmTEFs1 3′ UTRand/or the GmTEFs1 terminator to drive operably linked genes can bedeployed in many deciduous and evergreen timber species. Suchapplications are also within the scope of embodiments of thisdisclosure. These species include, but are not limited to; alder (Alnusspp.), ash (Fraxinus spp.), aspen and poplar species (Populus spp.),beech (Fagus spp.), birch (Betula spp.), cherry (Prunus spp.),eucalyptus (Eucalyptus spp.), hickory (Carya spp.), maple (Acer spp.),oak (Quercus spp.), and pine (Pinus spp.).

Use of GmTEFs1 promoter, the GmTEFs1 5′ UTR, the GmTEFs1 3′ UTR and/orthe GmTEFs1 terminator to drive operably linked genes can be deployed inornamental and fruit-bearing species. Such applications are also withinthe scope of embodiments of this disclosure. Examples include, but arenot limited to; rose (Rosa spp.), burning bush (Euonymus spp.), petunia(Petunia spp.), begonia (Begonia spp.), rhododendron (Rhododendronspp.), crabapple or apple (Malus spp.), pear (Pyrus spp.), peach (Prunusspp.), and marigolds (Tagetes spp.).

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A nucleic acid vector comprising a promoteroperably linked to a heterologous polynucleotide having a sequence of:a) a polylinker sequence; b) a non-Glycine max translation elongationfactor-1 alpha (non-GmTEFs1) heterologous coding sequence; or c) acombination of a) and b); wherein said promoter comprises apolynucleotide sequence that has at least 98% sequence identity with SEQID NO:2.
 2. The nucleic acid vector of claim 1, wherein said promoter is371 bp in length.
 3. The nucleic acid vector of claim 1, wherein saidpromoter consists of a polynucleotide sequence that has at least 98%sequence identity with SEQ ID NO:2.
 4. The nucleic acid vector of claim1, wherein said promoter is operably linked to a heterologous codingsequence.
 5. The nucleic acid vector of claim 4, wherein theheterologous coding sequence encodes a selectable marker protein, aninsecticidal resistance protein, a herbicide tolerance protein, anitrogen use efficiency protein, a water use efficiency protein, a smallRNA molecule, a nutritional quality protein, or a DNA binding protein.6. The nucleic acid vector of claim 1, further comprising a terminatorpolynucleotide sequence.
 7. The nucleic acid vector of claim 1, furthercomprising a 3′ untranslated polynucleotide sequence.
 8. The nucleicacid vector of claim 1, further comprising a 5′ untranslatedpolynucleotide sequence.
 9. The nucleic acid vector of claim 1, furthercomprising an intron sequence.
 10. The nucleic acid vector of claim 1,wherein said promoter has constitutive tissue expression.
 11. Atransgenic plant comprising a nucleic acid construct comprising apromoter operably linked to a heterologous coding sequence, wherein thepromoter comprises a polynucleotide sequence that has at least 98%sequence identity with SEQ ID NO:2.
 12. The transgenic plant of claim11, wherein said plant is selected from the group consisting of Zeamays, wheat, rice, sorghum, oats, rye, bananas, sugar cane, Glycine max,cotton, Arabidopsis, tobacco, sunflower, and canola.
 13. The transgenicplant of claim 12, wherein said plant is Glycine max.
 14. The transgenicplant of claim 11, wherein the nucleic acid construct is inserted intothe genome of said plant.
 15. The transgenic plant of claim 11, whereinthe nucleic acid construct further comprises a 3′ untranslated sequence.16. The transgenic plant of claim 11, wherein said heterologous codingsequence has constitutive tissue expression.
 17. The transgenic plant ofclaim 11, wherein said promoter is 371 bp in length.
 18. A method forproducing a transgenic plant cell, the method comprising the steps of:a) transforming a plant cell with a gene expression cassette comprisinga Glycine max translation elongation factor-1 alpha (GmTEFs1) promoter,wherein said promoter has at least 98% sequence identity with SEQ IDNO:2, operably linked to at least one polynucleotide sequence ofinterest; b) isolating the transformed plant cell comprising the geneexpression cassette; and, c) producing a transgenic plant cellcomprising the GmTEFs1 promoter operably linked to at least onepolynucleotide sequence of interest.
 19. A method for expressing apolynucleotide sequence of interest in a plant cell, the methodcomprising introducing into the plant cell a polynucleotide sequence ofinterest operably linked to a Glycine max translation elongationfactor-1 alpha (GmTEFs1) promoter, wherein said promoter has at least98% sequence identity with SEQ ID NO:2.